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PMC2662403

. we explore circadian and infradian rhythms in time estimation ( te ) , having shown that the stage of the former is essential for the assessment of the latter in the case of a half - year - long spectral component probably related to geomagnetics in human systolic blood pressure ( sbp ) ( tarquini et al 1997 ; halberg et al 2001a ; cornlissen et al 2002 ; sothern et al 2006 ) . we here ask whether changes with age in a mental function , such as the te of an 1-minute duration , also differ as a function of circadian stage . our test of mental functioning for precisely timed action is performed inexpensively , without the need for bulky instrumentation beyond a stopwatch . the test is quick and easily repeated , and may also tell us whether and , if so , when one aspect of subjective time what is particularly important , the circadian components here mapped could serve as a marker for interventions , therapeutic or other . te is a basic and critical feature and index of mental processes ( piaget 1946 ; fraisse 1963 ; geissler and reschke 1987 ) . a circadian rhythm characterizes several approaches to estimating 2 minutes under conditions of 24-hour synchronization with ( stephens and halberg 1965 ) or desynchronization from ( halberg et al 1965 ; siffre et al 1966 ; halberg and reinberg 1967 ; ghata et al 1968 ; halberg 1968 ; wever 1979 ) a societal routine . the circadian rhythm persists under conditions of isolation from society with a concomitant decrease of the ratio subject time / environmental time ( cf . such results extend earlier pioneering studies by macleod and roff ( 1935 ) and were confirmed and extended by follow - ups that also considered putative effects of cosmic factors ( hillman et al 1994 ; halberg et al 1998 ) and infradians ( sanchez de la pea et al 1989 ) , among others . whether or not a change in ratio of subjective / environmental time during isolation relates to the lengthening of the desynchronized circadian rhythm in 2-minute te and in other variables , noted in most humans studied thus far ( wever 1979 ) , remains to be investigated in a broader context ( fraisse 1963 ; geissler and reschke 1987 ; glicksohn 1992 ; halberg et al 2001a ; eagleman et al 2005 ) . for example , when the subject of this paper ( rbs ) spent 19 days in social isolation near the beginning of his 40-year self - measurement project , the period of his circadian rhythm in 2-minute time estimation increased ~4% , from 24.0 to 24.9 hours , while the 24-hour mean increased about 22%25% , from about 150 s to 188 s ( unpublished observations [ cf . halberg et al 1972 ] ) . both a decrease in circadian amplitude during isolation and an increase in circadian amplitude after isolation are seen in figure 2c for the rhythm in core temperature ( top ) and 2-minute estimation ( bottom ) , along with a delaying drift of the acrophases due to a lengthening of period . hypothesized that the more varied the sensory environment ( ie , perceptual overload versus perceptual deprivation ) , the longer the time that is verbally estimated , and the shorter the time estimation obtained by the method of production . his data on 96 subjects exposed to various combinations of auditory and visual stimulation gathered by the latter method agreed , but not those with the former method . he concluded that visual and auditory stimulation interact in their production of an altered sensory environment , and that the rate of functioning of the cognitive timer is environment dependent . eagleman and colleagues ( 2005 ) review new discoveries in psychophysics , electrophysiology , imaging , and computational modeling [ that ] are contributing to an emerging picture of how the brain processes , learns , and perceives time , emphasizing that most of the actions our brains perform on a daily basis , such as perceiving , speaking , and driving a car , require timing on the scale of tens to hundreds of milliseconds . sothern and colleagues ( 2007 ) most recently found a near - transyearly period , ( 1.00 year < [ ci { 95% confidence interval } ] < + ci ] < 1.20 years ) , and several far - transyearly s ( 1.2 years [ ci ] < [ + ci ] < 1.9 years ) in 1-minute te ( about 5 times a day for 35 years ) as a magnetoperiodism , with a differential congruence of these near- and far - transyear periods with the periods of cycles in the solar wind s speed and geomagnetics . here we focus on the photic circadian and other rhythm - stage dependence of changes in 1-minute te and on any effects of age . for 3.5 decades , 1 minute was estimated by counting two to seven times daily ( on the average about 5 times / day ) , by a clinically healthy man ( rbs ) , who started estimating 2 minutes by counting at ~20.5 years of age in 1967 . in view of differences between te of 2 minutes , performed at the start of monitoring for ~3 years and subsequent 1-minute te continued a year later for 3.5 decades , only data from the longer 35-year record ( of 1-minute te ) are here considered . data were analyzed spectrally , by the extended linear - nonlinear cosinor ( halberg 1980 ; cornlissen and halberg 2005 ; refinetti et al 2007 ) with parameter tests ( bingham et al 1982 ; cf . halberg et al 2003a ) and by comparing the first and the last year s original data in 8 time - of - day bins of 3 hours each with student s t - test and all 8 timepoints by a paired t - test . in figure 3 , the periodogram of rbs s data is plotted linearly in frequency , yet with periods given on top for each half of the figure , the upper half summarizing results at 25 years of age , and the lower one at 60 years of age . the original ordinate at the bottom of each , the upper and lower halves , is given on the right . in the insert of each half , the ordinate is truncated to prevent the dominating circadian from obscuring lower - than - circadian frequencies ( infradians ) . the inserts show that with advancing age ( lower vs upper half ) , some frequencies differ in prominence . while a circaseptan and a circasemiseptan component are detected with statistical significance at both ages , a 30-day component is detected prominently only at the younger age . at 60 years of age , a 1-year component s prominence is replaced by that of a half - yearly component , probably of geomagnetic ( nonphotic ) origin ( cornlissen et al 2002 ) . figure 4 provides a highly qualified yet clear answer to the question whether there is an acceleration of the subjective minute with age in rbs . this figure stacks data in two chronograms along the 24-hour scale , each summarizing a year s data 35 years apart , at 25 vs 60 years of age , respectively . without extrapolating to other subjects ( halberg et al 1981 ) and/or to the intervening years , admittedly great limitations , both the ( sparsely documented ) maxima and some of the ( solidly documented ) minima ( numbers of te at each time are given in parentheses ) are numerically lower during year 35 than during the first year ( figure 4 ) , ie , at 60 vs 25 years of age , suggesting acceleration . figure 4 shows further that even if overall the time - structure - adjusted mean ( mesor ) is numerically lower at an advanced age , the demonstration of an age change is not possible on this basis ( p = 0.300 ) . a circadian stage - dependence in figure 4 is found by student t - tests that show an aging effect in rbs to be not demonstrable at certain circadian stages , while it is statistically highly significant at other times of day ; each result , that around 10:30 vs that at 19:30 , is documented with relatively ample data . these results , albeit amply documented for rbs , can not be extrapolated to others . in another case , hl , a physician with aldosteronism , the estimation of 2 minutes took longer at 63 years of age than it did at 53 years , as if time passed more slowly in this particular elderly man ( halberg et al 1981 ) . while in hl the change during a decade seems to be linear , it was nonlinear over several decades in the yearly and transyearly amplitudes of rbs heart rates ( hr ) ( sothern et al 2004 ) and what it was in te remains to be assessed . assessed for rbs and hl may perhaps be reconciled , at least in part , by the presence of low - frequency cycles . in the case of rbs , even decade - long records may thus exhibit either an increasing or a decreasing trend depending on when during this about 26-year cycle monitoring took place . a similar situation was encountered in a 15-year record of urinary excretion of 17-ketosteroids characterized by an about 10-year cycle ( halberg et al 2001b ) . in the case of rbs , subjective time elapsed during the 19 days of isolation was shorter by 17.5 hours or ~4% . this is contrasted with 2-minute time estimation by rbs that was increased by 25% from ~150 to ~188s , suggesting a dissociation between short - term and long - term time perception , the difference along the two time scales possibly accounted for , at least in part , by the rest - activity cycle , which provides an important clue of elapsed time along the scale of days . differential congruence of periods ( s ) in helio- and/or geomagnetics and in human psychophysiology is also documented for rbs assessed ( by overlying or overlapping 95% confidence intervals ) of s found in a mental function ( 1-minute te ) and in sbp and hr in about 4 decades of around - the - clock self - measurements ( sothern et al 2007 ) . a combination of the extended cosinor by nonlinear analyses complemented by a population - mean cosinor summarizes the same series analyzed in consecutive subsections . there are corresponding s in te , sbp and hr themselves and in the spectrum of solar wind speed and in geomagnetics ( aa index ) for several orderingly statistically significant peaks between trial s of 2.0 and 0.7 years ; this congruence differs among variables and s involved ; it is differential . 5 of 8 sbp peaks are congruent with the solar wind in the spectral window examined at s different from a calendar year , as are 3 of the 4 sbp peaks that are congruent with geomagnetics . congruence of sbp with magnetoperiodisms occurs in the presence of a large photoperiodic annual component in sbp . congruence of s with solar wind is 3 out of 7 for hr and 5 out of 10 for te , and with geomagnetics it is 2 out of 7 for hr and 4 out of 10 for te . different physiological variables and even the same variable s different spectral components may be influenced by different environmental s . while no congruence in all 5 variables was seen , all 3 psychophysiological variables were congruent with richardson s ~1.3-year oscillation in solar wind speed during the span of rbs s data . congruence among an as - yet undefined fraction of s in other physical and biological variables monitored by rbs and others is also differential ; it is not conclusive in itself but is desirable for a follow - up by superposed epochs and by comparison of time courses of s in and around us , whenever rendered possible due to removal and replacement of intermittent s by the sun and earth . magnetoperiodism coexists and competes with photoperiodism to influence dynamics in the same individual and can dominate in te in the range of near - transyears , while photoperiodism dominates in the circulation of the same individual in both the para - annual and circadian spectral domains . in any event , figure 4 suffices to infer that in the estimation of subjective time different aspects of the body s temporal make - up interact in a circadian stage - dependent way in the case of rbs ; hence , age - dependent acceleration in estimation of 1 minute is best specified as to circadian time and probably as to other rhythms stages so that a different outcome , such as an acceleration vs no age effect , and in another case ( hl ) opposite inferences , can be taken at face value . on the positive side , the recognition of cycles , including non - photic ones such as transyears , in time estimation that differ from the photic day and year opens new perspectives of how a mental function can be influenced by the solar wind s cyclicities that may have entered the genome , as circadians clearly did ( touitou and haus 1992 ; halberg et al 2003b ; koukkari and sothern 2006 ; refinetti 2006 ) . the importance of nonphotic cycles beyond those in the estimation of 1 minute is evident from those influencing sudden death ( halberg et al 2006 ) , suicide ( gordon and berk 2003 ; berk et al 2006 ; cornlissen and halberg 2006 ; shumilov et al 2008 ; chernouss et al pers comm ) , terrorism ( cornlissen et al 2007 ; grigoryev and vladimirskii 2007 ; halberg et al 2007 ; wendt 2007 ) , and psychophysiology more generally ( persinger 1987 ; oraevskii et al 1998 ; breus et al 2002 ; palmer et al 2006 ) . in a follow - up study focusing on the years intervening between ages 25 and 60 , opposite effects at different circadian test times could be validated with statistical significance . these findings extend the scope of the comparison of the first and the last year of a 36-year record , which latter sufficed to show that one can have different results concerning aging , depending on when along the scale of a day , 1 minute is estimated by counting . a 1-minute duration passed faster at 60 vs 25 years of age at some circadian stages , eg , between 09:00 and 12:00 , while at other circadian stages no difference or an opposite trend between 18:00 and 21:00 ( r = 0.400 , p = 0.010 ) was detected . if one examines the change with age of hormones in women , one finds an increase ( p < 0.001 ) for lh and a decrease for e2 ( p < 0.001 ) ( halberg et al 1981 ) . decreases are also found for aldosterone , melatonin and in particular for dehydroepiandrosterone sulfate ( halberg et al 1981 ; cornlissen et al 2000 ) . accordingly , a change in amplitude with age , ceteris paribus , will involve necessarily as a function of age opposite results at the circadian peak versus trough : an increase in amplitude will have a corresponding increase at the time of circadian peak and a decrease at the time of circadian trough and vice versa in the case of a decrease in amplitude there will be a decrease at the time of circadian peak and an increase at the circadian trough . this applies to circadian rhythms and has been documented in halberg and colleagues ( 1981 ) and cornlissen and colleagues ( 2000 ) . similar effects will apply to any other cycle changing with age and controversies will be unavoidable even if the time of day is fixed but chosen differently in various studies , according to investigators convenience . a further complication is also relevant to studies on aging between just reaching maturity and at 60 years of age . a plethora of aeolian cycles , figure 5 , are characteristic of many functions monitored not only by rbs but also on over a dozen other subjects of both genders with decades - long time series . many of these spectral components are of non - photic origin , coexisting and competing with , and sometimes replacing the calendar - year component . these cycles constitute pertinent control information and are also of interest in their own right , since some of them are associated with suicide ( cornlissen and halberg 2006 ) and sudden cardiac death ( halberg et al 2006 ) . aging can be studied without or with chronomics : so it appears to be the case today . but if opposite inferences are drawn at different circadian stages , as documented herein and earlier for another subject , sbs ( sothern et al 2006 ) , it seems possible that we may generalize and consider requiring the time of day as an indication in all studies of human aging . this seems indicated based on evidence available today , so that controversies are avoided and expensive long - term studies must not be repeated . moreover , an indication of calendar date in any and all studies of aging seems to be similarly required , since some of the cycles documented here and elsewhere can be signatures of environmental periodicities , which latter are recorded by governmental agencies and can thus be consulted and used for computing reference cycles . for human mental function , there is a saying : use it or lose it . when to use it , to avoid the loss of cognitive function , and when to treat , is already extensively documented for circadians and may also apply to infradians , for instance , since negative affect is primarily circaseptan periodic , while positive affect is dominantly circadian ( cornlissen et al 2005 ) , as shown herein for mental functioning by the proxy of time estimation . the amplitudes of the new rhythms mapped may be small , but small amplitudes are compatible with large and important associations ( halberg et al 2003c ) . the circadian amplitude in the mental function mapped along the age scale remains large and may serve immediately for timing intervention ; timing , like dosing , has demonstrated , albeit as yet not routinely utilized , therapeutic benefit .

backgroundcircadian rhythm stage affects many outcomes , including those of mental aging.methodsestimations of 1 minute ~5 times / day for a year , 25 years apart , by a healthy male biomedical scientist ( rbs ) , are analyzed by the extended cosinor.resultscycles of a half - week , a week , ~30 days , a half - year and a year , in self - assessed 1-minute estimation by rbs between 25 and 60 years of age in health , are mapped for the first time , compared and opposite effects are found . for rbs at 60 vs at 25 years of age , it takes less time in the morning around 10:30 ( p < 0.001 ) , but not in the evening around 19:30 ( p = 0.956 ) , to estimate 1 minute.discussionduring the intervening decades , the time of estimating 1 minute differed greatly , dependent on circadian stage , being a linear decrease in the morning and increase in the evening , the latter modulated by a ~33.6-year cycle.conclusioncircadian and infradian rhythm mapping is essential for a scrutiny of effects of aging . a ~30-day and a circannual component apparent at 25 years of age are not found later ; cycles longer than a year are detected . rhythm stages await tests as markers for timing therapy in disease .

Introduction Background Subject and methods Results Discussion Conclusion

PMC4561316

the immediate early genes ( iegs ) were first described in viruses and then identified in various cell lines . the iegs are transcribed following a variety of stimulations such as growth factors , hormones , and cytokines in a protein synthesis - independent fashion . their relevance for the study in adult neuronal plasticity was first brought to light in 1987 , when it was shown that c - fos , a protooncogene that is also a transcription factor , was rapidly transcribed in neurons following seizures . a couple of years later , another transcription factor , zif268 , was identified ; it was expressed after plasticity inducing treatments such as maximal electroconvulsive shocks and long - term potentiation ( ltp ) . it has also been demonstrated that zif268 transcription is dependent on n - methyl - d - aspartate ( nmda ) receptors activity , suggesting a functional link between these receptors and iegs in the process of synaptic plasticity [ 3 , 4 ] . in the following years , paul worley and collaborators undertook the task of identifying iegs whose products were directly involved in modifying cellular function , rather than transcription factors with a presumably indirect role . this gave rise to the discovery of a whole new set of effector iegs : the cox-2 an enzyme involved in lipid metabolism that was later shown to be involved in long - term plasticity and memory , homer1a , a scaffold protein that interacts with metabotropic glutamatergic receptors and modulates intracellular calcium signaling , and activity - regulated cytoskeletal - associated protein ( arc ) , a protein involved in synaptic remodeling and plasticity [ 912 ] . these ieg products appeared as excellent candidates for proteins whose ongoing synthesis is essential for ltm to occur . however , an obvious intriguing question remained in how do proteins , newly synthesized in the soma , become associated with potentiated synapses ? in order to explain that question , the concept of synaptic tagging was introduced . synaptic tagging is the idea that a translation - independent molecular mark must be established at potentiated synapses in order to provide input specificity for long - term , protein synthesis - dependent plasticity mechanisms [ 13 , 14 ] . with arc being a candidate for plasticity related proteins recruited by putative synaptic tags after ltp - inducing stimulation of the perforant path , arc mrna was shown to accumulate specifically in the medial molecular layer of the dentate gyrus ( dg ) , that is , the dendritic region that received the bulk of the stimulation during this procedure [ 15 , 16 ] . importantly , this phenomenon was later explained by the dendritic transport of its mrna , which also was obliterated by nmda receptors antagonism [ 1518 ] . further insight on the involvement of arc in memory formation was gained when researchers examined the dynamics of arc mrna in the hippocampal network after exploration of a novel environment . that is , after 5 min of spatial exploration arc mrna was reliably detected in the nuclei of activated cells of the hippocampus and cortex . interestingly , 2530 minutes later , the percentage of cells expressing arc mrna in the nucleus was comparable to that of control animals , as the transcript already traveled to the cytoplasm where it was reliably detected [ 19 , 20 ] . this kinetics of arc mrna combined with the specificity to physiologic stimuli [ 19 , 21 ] has allowed the design of a method combining in situ hybridization and confocal microscopy to detect large neuronal populations activated by two or even three distinct behavioral epochs [ 22 , 23 ] . this tool , termed catfish ( for cellular compartment analysis of temporal activity by fluorescence in situ hybridization ) , has helped to advance our understanding of the neuronal circuit underlying memory storage in a variety of behavioral paradigms . the catfish technique allowed demonstrating that the population of cells expressing arc during a subsequent exposure to the same environment highly overlaps with those expressing the mrna during the first period . however , when the two behavioral epochs consisted in two strikingly distinct environments , the populations of cells expressing arc were shown to be statistically independent . noteworthy , in vivo single unit recordings have shown that , during exploratory behavior in rats , ~18% of ca3 and ~40% of ca1 neurons show place field activity . interestingly , it was discovered that a similar proportion of neurons express arc mrna in the nucleus . thus , since these place cells are widely believed to store contextually relevant information , this further pointed to a role in arc in declarative memory that was consistent with the conjunctive trace model . accordingly , it was demonstrated that acute intrahippocampal inhibition of arc translation during the hours following acquisition impaired ltm of a spatial navigation task . a more recent study by the same group showed that inactivation of the medial septum , a treatment known to impair hippocampus - dependent learning and memory , abolishes behaviorally induced arc expression in this region . importantly medial septum inactivation is known to spare location specific firing in ca1 place cells . these findings thus strongly suggest that arc expressing neurons represent a memory storing engram rather than neuronal activity per se and further strengthen the rationale behind mapping arc gene expression in neuronal networks during behavior . importantly , arc expression mapping has been helpful to visualize memory storing neuronal networks not only in the hippocampus but also in several cortical and subcortical regions under a wide variety of behavioral paradigms . for example , some researchers took advantage of the conditioned taste aversion ( cta ) task in which a strong associative memory is formed even if the conditioned stimulus ( a novel taste ) and the unconditioned stimulus ( postingestive induced malaise ) are presented 25 min or even more apart . this considerable time lapse between stimuli allowed the authors to perform a catfish design allowing visualization of the convergence of a conditioned stimulus with the unconditioned stimulus onto single neurons in the basolateral amygdala . indeed , some amygdala neurons were activated by both stimuli ( had both nuclear and cytoplasmic arc mrna ) . however , when the stimuli presentation was reversed , that is , the licl injection was first and then the saccharin solution was presented after 25 min , the proportion of double stained amygdala neurons was dramatically decreased . these results strongly suggested that the observed convergence in the forward conditioning represented associative learning rather than mere overlap in the neuronal response . later , inspired by this study , another group of researchers used the conditioned odor preference task and showed that neurons of basolateral amygdala learned to associate an odor with an appetitive taste outcome , as a repeated convergence of taste and odor induced arc mrna increments after several days of pairing the smell with the taste . a similar phenomenon was observed in the insular cortex by another group ; they showed that an odor cue associated with a taste was as efficient at driving ieg expression in insular cortex neurons as the taste itself . moreover , in this study it was found that when the same taste was presented twice , it tended to induce ieg transcription ( arc and homer1a ) in the same subset of neurons in the insular cortex , just as it occurred in the hippocampus after repeated exploration of the same environment . as mentioned earlier , intranuclear foci of immature arc can be detected 2 to 5 min after exposing rats to an open field . if the groups of neurons that express arc after information encoding were memory storing networks , one would expect that changes in synaptic activity would play a major role in this fast and discrete arc expression . efforts were thus deployed at identifying the precise cascade of events , from the synapse to the nucleus , that give rise to arc expression . a role of putative memory - associated signaling pathways was early suspected and , accordingly , it was found that depolarization - induced arc in neurons was dependent on intracellular calcium influx and activation of camp dependent protein kinase and extracellular signal regulated kinase signaling pathways . later , another group showed that glutamate release at excitatory synapses induces rapid arc mrna transcription in hippocampal neurons by a mechanism that depends on the transcription factor myocyte enhancer factor type 2 activation . however , the effects on arc expression obtained in these studies were rather modest considering the robust increase observed under physiological conditions [ 16 , 19 , 27 ] . a more recent study further sought to identify highly preserved cis - acting elements in the arc promoter that could account for the very tight and dramatic activity - dependent increase of arc transcription reported in earlier studies . screening more distal parts of the arc promoter ( ~7 kb ) they found a ~100 bp element that was sufficient to replicate the full extent of arc 's activity - dependent induction ( ~150 fold increase ) after periods of intense activity in vitro and coined this element , importantly blocking -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) and nmda receptors abolished sare - induced transcription . noteworthy , regions within the sare element matched consensus binding sequences for cyclic adenosine monophosphate responding element binding protein , serum response factor , and myocyte enhancer factor type 2 ( creb , srf , and mef2 , resp . ) , three transcription factors strongly involved in neural plasticity . therefore , this element provided a mechanism by which arc transcript can be strongly induced , specifically by synaptic activity . in addition to the activation of the sare element , a mechanism that ensures rapid synaptic activity - dependent arc transcription was recently unveiled , which resides in stalled rna polymerase ii at the transcription initiation starts of arc promoter . poised polymerase , along with active chromatin marks and preloaded transcription factors , provides a mechanism by which an activity induced signal can bypass the time - consuming process of transcription initiation and release rna polymerase ii for active transcription . interestingly , interfering with rna polymerase stalling affected rapid induction of arc but spared delayed ieg such as early growth response protein 3 . thus , these new findings on the molecular events that underlie arc transcription help to explain how it can exert its function in behaviorally activated cells , in a fast and specific manner . experiments aimed at uncovering the role of arc in synaptic plasticity at molecular and cellular levels showed that , in dendritic spines , arc associates with the endocytic machinery , interacting with dynamin and endophilin 2/3 , components of the clathrin - dependent endocytic machinery , thus enhancing ampa receptors endocytosis . arc is strongly induced in neurons where its protein downregulates surface ampa receptors after periods of increased neural activity . thus activity induced arc has a role in homeostatic synaptic scaling [ 44 , 45 ] , a non - hebbian form of plasticity that serves to shift back neural excitability to physiological range , while preserving the relative change in individual synapses induced by hebbian forms of plasticity , such as ltp [ 4648 ] . moreover , rapid dendritic translation of constitutive arc mrna has been shown to underlie metabotropic glutamate receptors- ( mglur- ) dependent long - term depression ( ltd ) through arc - dependent ampa receptors endocytosis . the role of arc in the cell - wide weakening of glutamatergic synapses seemed counterintuitive , based on abundant evidence showing accumulation of both arc mrna and protein in potentiated dendritic regions [ 15 , 29 ] , as well as its requirement for ltp maintenance [ 25 , 50 ] . however , groundbreaking new evidence was brought to light in a recent paper by hiroyuki okuno and collaborators that reconciled the role for arc in synapse - specific homeostatic plasticity and synaptic tagging . the authors first used a yeast two - hybrid screening to identify protein partners binding to arc and identified an interaction with calcium / calmodulin - dependent protein kinase ii ( camkii ) . this interaction was found to be stronger in the absence of the ca / cam complex , suggesting a preferential interaction with the inactive form of the kinase . moreover , after reliably and robustly inducing global arc expression in neurons , the authors examined the effect of locally suppressing synaptic activity at single presynaptic sites . strikingly , this treatment increased arc accumulation at the inactivated synapses and , there , arc was shown to diminish surface ampa receptor glur1 subunit content . together , these results show that after periods of increased activity that induce robust arc expression in neurons , it specifically accumulates at inactive spines by interacting with the inactive form of camkii , that is not bound to calmodulin , enabling what was termed inverse synaptic tagging . the role of arc , therefore , appears not only to scale down neural excitability after hebbian synaptic modifications but also to crucially increase the contrast between potentiated and nonpotentiated synapses . as mentioned previously , global arc mrna increments have consistently been observed at recently activated dendritic regions [ 22 , 29 ] . it is probable that , under the settings used in these studies and as acknowledged by the authors , ltp occurs only in a subset of the stimulated synapses , as is also thought to occur during learning . conceivably , accumulation of arc mrna at activated dendrites or dendritic zones could provide a mechanism where inactive synapses in the vicinity of recently potentiated ones swiftly recruit massive amounts of arc protein for synaptic depression to occur at these sites . however , as we shall see in the next section , a wealth of in vivo evidence also argues in favor of a distinct and specific role for de novo arc translation in ltp consolidation at recently stimulated synapses . a growing body of evidence in favor of a direct role for arc in synapse strengthening , at least under certain conditions , has recently received further support . first to be mentioned is that de novo arc protein synthesis was soon shown to be required in vivo for the maintenance phase of ltp of the perforant path . later studies in arc knockout mice confirmed a role for arc in both ltd and ltp . specifically , ltp induction was shown to be enhanced , while the maintenance phase was abolished in both perforant path and schaffer collateral pathways , in agreement with the previous findings . however , the strongest piece of evidence in favor of ltp consolidation appears to come from studies using perforant path stimulation of dg 's granule cells . noteworthy and contrary to what happens in hippocampal pyramidal cells where both ltp and novel environment exploration induce a robust and temporally discrete wave of arc expression , a more gradual and sustained increase of arc mrna and protein appear to be produced by these procedures in dg 's granule cells [ 33 , 5456 ] . importantly , local infusions of arc asodns at 2 h following in vivo high frequency stimulation of the perforant path abolished ltp maintenance and impaired f - actin polymerization and cofilin phosphorylation , molecular events that are thought to underlie learning - induced structural plasticity [ 30 , 55 , 57 ] . most strikingly , treatment with the actin stabilizing drug jasplakinolide , between ltp induction and arc asodns treatment , abolished the deleterious effects of arc translation inhibition on ltp maintenance . these results strongly suggest that arc 's role in dg ltp consolidation rests in its ability to stabilize recently polymerized actin filaments . finally , arc asodns infusions before ltp induction with high frequency stimulation or bdnf infusions prevented ltp expression indicating that arc translation was required for early ltp expression as well as maintenance . recently mechanistically distinct rounds of translation that depended on sustained mnk activation through bdnf signaling were shown to underlie dg - ltp . infusions of bdnf scavenger trkb - fc or mnk inhibition brought field evoked postsynaptic potentials as well as arc protein translation back to baseline . all in all , a very strong case can now be made for a direct role of arc in dg - ltp . as observed before [ 59 , 60 ] this however , nothing supports the a priori principle that arc 's function should be similar in every studied cell type . in fact , its role may differ between pyramidal and granule cells , as it was recently proposed for bdnf 's . this possibility should draw serious attention given that , as mentioned earlier , it is now demonstrated that arc expression kinetics in granular and pyramidal cells differ dramatically . further , still little attention has been paid to possible posttranslational modifications to arc protein as it was observed in an earlier paper . however , possible phosphorylation sites for pkc and camkii have been identified since the protein 's discovery . as pointed out recently , and regardless of the experimental settings or cell type , the bulk of arc protein observed in principal activated cell appears in the perinuclear cytoplasm , where its function remains obscure although it is now established that at least part of it is shuttled to the nucleus . arc protein was first detected in the cell nucleus of cultured hippocampal neurons in association with promyelocytic leukemia bodies ( pml ) , which are putative sites of transcriptional regulation . consistently , a more recent study further showed that stimulating dg granular cells for prolonged periods , with brain - derived neurotrophic factor or bicuculline , induced a gradual targeting of arc to the nucleus that reaches peak levels at 8 h. there , arc promotes the assembly of nuclear pml bodies , which , in turn , negatively regulate the transcription of the ampa receptor subunit glur1 . importantly also , nuclear localization was also observed after exposure to a novel environment not only in the granular cells of the dg but also in hippocampal ca1 and ca3 regions and in the somatosensory cortex . importantly , the kinetics of arc accumulation to the nucleus varied depending on the brain region and cell type . these findings thus provide an additional , cell - wide mechanism by which arc promotes homeostatic plasticity , after prolonged periods of synaptic activity . all in all , arc accomplishes distinct functions depending on its interaction partners and the time course of its accumulation . another interesting observation is that the arc - dependent downregulation of surface ampa receptors appears to be specific to certain dendritic spines , depending on their morphological characteristics . dendritic spines can indeed be classified in distinct categories according to their shape , size , and structure , which are correlated with synaptic strength , motility , and structural plasticity . mushroom spines are larger , are much more stable , and have a greater amount of ampa receptors than thin spines that are also much more labile and dynamic . for these reasons , mushroom spines have been referred to as memory spines , whereas thin spines are the putative learning spines . in agreement , further , arc knockout mice have increased seizure sensitivity and epileptiform activity as measured with electroencephalogram , whereas arc / neurons have decreased spine density but , crucially , increased spine width . these findings confirmed a role for arc in homeostatic synaptic scaling and global network stability . arc protein targeting at synapses tagged as inactive would diminish unspecific noise and allow nearby potentiated arc - negative thin spines to stand out and eventually become memory spines . conceivably , synaptic potentiation and spine growth could be the default mechanism that occurs in behaviorally activated cells ; it well could be that synaptic inactivity could be the trigger that confers specificity . alternatively , distinct , yet complementary , mechanisms could occur at active synapse that would further increase the contrast between potentiated and unpotentiated synaptic networks ( see figure 1 ) . given the synaptic localization of arc protein , its tight activity dependence , and its striking effects on synaptic function , efforts have been deployed to uncover its possible role at distinct phases of the process of learning and memory . the generation of arc knockout mice revealed a role for arc in long - term but not short - term memory in a variety of tasks , including object recognition memory and amygdala - dependent tasks , such as conditioned taste aversion and fear conditioning , showing that learning per se is unaffected in these mice . furthermore , the formation of long - term spatial memory as assessed by morris water maze task was impaired ; arc knockout mice were slower learners , formed a less precise memory , and , interestingly , showed less behavioral flexibility as they took longer to relearn a new position of the target platform . these results in the arc knockout mice were in accordance with the seminal paper of guzowski and collaborators mentioned earlier that found impaired long - term spatial memory after acute arc translation blocking , therefore providing a first causal link between de novo arc protein synthesis and ltm consolidation . a requirement for de novo arc protein expression for consolidation and reconsolidation processes was later unveiled in a great variety of learning and memory paradigms . for example , in the amygdala , a key structure involved in the storage of the emotional contingency related to a context or a stimulus [ 65 , 66 ] , it was shown that administration of arc antisense oligodeoxynucleotides ( asodns ) before training the animals in a pavlovian fear conditioning task affects its consolidation . furthermore arc asodns administration 90 min before reactivation of the same task in the lateral amygdala impaired reconsolidation of this task . on the other hand , infusions of arc asodn in the basolateral amygdala 3 h before extinction of a contextual fear conditioning task impaired shifting of the emotional component of the context from aversive to safe . similarly , the importance of de novo arc translation for ltm formation was also demonstrated in the neocortex , in both associative and nonassociative memory paradigms . for example , posttraining administration of arc asodns in the cingulate cortex was reported to disrupt ltm formation in an inhibitory avoidance paradigm . in our lab , we showed that inhibiting arc protein synthesis in the insular cortex prevents familiarization with a safe taste and hinders the hedonic shifting of a taste from aversive to safe during extinction of conditioned taste aversion ( cta ) ( guzman - ramos et al . these data demonstrate that de novo arc protein expression in critical mammalian forebrain structures plays an essential role in ltm formation . therefore , while some factors have been identified that specifically operate in either consolidation or reconsolidation , this does not seem to be the case of arc which synthesis appears to be required indistinctively for both processes [ 71 , 72 ] . these studies indicate that de novo arc protein synthesis in the participating brain structures seems to be required for both processes . furthermore , they are in agreement with observations by ours and other groups showing that arc protein expression increased once a behaviorally relevant stimulus becomes familiar ( see below ) . in many tasks , the requirement for nmda receptors activity in order to consolidate ltm has been clearly established . a functional link between nmda receptors activation and iegs expression has long been suspected to underlie specific synaptic modifications that are essential for the establishment of a stable memory trace . as a matter of fact , inhibition of nmda receptors is known to hinder activity - dependent arc mrna expression , localization at activated dendritic sites , and degradation [ 9 , 15 , 29 ] . however , a direct involvement of arc - nmdar interdependency in vivo during learning was not tested until recently . to this matter , one group used contextual fear conditioning , a task known to involve nmda receptors - dependent plasticity mechanisms in the hippocampus , and showed an increase in arc protein accumulation in the hippocampus at 1 h after acquisition that was blocked by nmda receptor antagonist apv . furthermore , pretraining infusions of arc asodns impaired consolidation but spared acquisition of contextual fear conditioning tasks . these results clearly point to a role of nmda receptors - dependent arc synthesis in the hippocampus in the formation of a long - term contextual fear memory . yet a more recent study sought to determine whether this arc - nmda receptors synergy was also involved in memory retrieval of a familiar task . moreover , nmda receptors activity upon retrieval proved to be essential for contextual memory maintenance as the increased locomotion upon subsequent context exposure was attenuated in rats treated with nmda receptors antagonist apv . these findings bring to light further support for the idea that retrieval memories , even well - consolidated ones , place the involved circuits in a labile state that require further nmda receptors - dependent arc protein synthesis for their stabilization . in the next section we will discuss recent findings regarding the phenomenon of retrieval - induced plasticity mechanisms , with a focus on arc , and their possible role in memory stabilization and persistence . hebb 's second postulate stipulated that , in addition to feed - forward synaptic strengthening , reverberation of neural ensembles must occur in order to form a temporary unit of memory storage . these ensembles of neurons facilitate coincidence detection of upcoming sensorial information by integrating information from temporally related , but spatially segregated , neural activity . in recent years , several lines of experimental evidence have brought support and refined hebb 's proposal . in our lab , we examined putative offline reactivations after associative learning at the level of neurochemical extracellular changes , using the conditioned taste aversion paradigm . first , we found a significant increase of dopamine levels in the insular cortex during the ingestion of a novel saccharin solution . second , intraperitoneal injection of licl , used as an unconditioned stimulus in this task , was shown by itself to produce a swift increment of glutamate release in this same structure . strikingly , however , a delayed , concomitant release of dopamine and glutamate was observed in the insular cortex that was abolished by reversible inactivation of the amygdala , another structure involved in long - term conditioned taste aversion memory formation [ 78 , 79 ] ( interestingly , similar results with the neurotransmitters norepinephrine and glutamate were observed in the amygdala ) . arguably , the concomitant dopamine and glutamate release we reported in the insular cortex could precede and be required for more enduring forms of synaptic plasticity in these regions that were reported by our group and others [ 37 , 81 , 82 ] . currently , the most widely accepted model accounting for ltm formation stipulates that learning induces morphological and functional modifications at activated synapses and subsequent learning - dependent protein synthesis allowing stabilization of these modifications , so that the newly strengthened synaptic networks become stored for days to months , a phenomenon that was termed synaptic consolidation . recently , an emerging subfield of neuroepigenetics , the study of the role of epigenetics mechanisms in adult neurons , has recently unveiled possible mechanisms by which synapse - specific changes induced by learning could remain permanently . in this regard , the molecular mechanisms of memory maintenance focused on the cell 's nucleus have been proposed ; that is , covalent modifications of the dna are the ultimate biochemical event that could store information permanently . notably , this phenomenon could work in parallel with wider distribution of the memory trace through cortical networks in order to further stabilize memories . the concept of epigenetics refers to changes in gene transcription through modulation of chromatin , which are not brought by changes in dna sequence . there are two possible ways in which these modulations of chromatin could play a role in memory storage . on one hand , stable chromatin modifications can interdigitate with synaptic tags in order to participate in and maintain synapse - specific changes . another possibility could be that neuroepigenetic mechanisms since they operate at a cell - wide level could induce metaplasticity in selected populations of neurons so that the tuning up or down of specific synapses would be permanently facilitated . importantly , epigenetic modifications at promoter sites of various plasticity related proteins , including arc , have recently been described [ 88 , 89 ] . as mentioned earlier arc - dependent ampa receptors endocytosis operates both at a synapses specific level , through synaptic inactivity - dependent interaction with camkii [ 51 , 52 ] , and in a cell - wide fashion , through downregulation of glur1 transcription . interestingly , methylation of the arc promoter that correlated in time with a decrease in arc protein below basal levels has been reported at 24 h after the induction of electroconvulsive seizures . also , aberrant changes in arc promoter methylation in hippocampal neurons have been suggested to play a role in age - related cognitive decline . methylation , a putative gene silencing signal , is arguably the most stable epigenetic modification and could serve to maintain changes in gene expression dynamics induced by memory consolidation . indeed methylation of memory suppressing gene calcineurin was induced in the frontal cortex after contextual fear conditioning and persisted for at least 30 days . further , interfering with the enzymes responsible for maintaining methylation on cytosine residues , on the 30th day after conditioning , significantly impaired retention of the task . on the other hand , knocking - down of tet1 , an enzyme that promotes dna demethylation , is associated with decreased expression of synaptic plasticity related , putative memory enhancer genes nasp4 , c - fos , egr2 , and arc as well as abnormally enhanced ltd and impaired memory extinction . this suggests that the methylation rate of these genes affects the effectiveness of subsequent plasticity inducing events , thus modulating their ability to update consolidated memories upon reactivation . taken together , these findings provide crucial insights on the role of chromatin modifications in long - term memory persistence and will undoubtedly set the basis for important new discoveries in this field , in the years to come . meanwhile , they might also provide a rationale behind the robust arc and other plasticity related proteins ' expression that is observed after retrieval of even some characteristics of arc expression during ongoing behavioral experience , especially at the posttranscriptional level , are somewhat counterintuitive , given its role in memory consolidation . in fact under many setups it has been observed that arc mrna and protein are still expressed at high levels after the animal experiences an already familiarized context or stimulus . further , under some circumstances , exposure to a familiar behavioral stimulation induces even greater arc expression . in a recent study exploring arc mrna expression dynamics in hippocampal subfields after running around a track in a novel context , optimal arc expression was observed in ca3 after a rat ran around a track a single time ; no further increment was observed when the animal ran several times around the same track or when it ran several times for four consecutive days . in ca1 , on the other hand , the greater proportion of arc expressing cells was observed in the condition where the animal ran several times around the track for the fourth consecutive day in the same context . first of all , these data provided compelling support at the molecular level for a role of ca3 in the fast encoding and subsequent storage of a novel context . further , it provided evidence that behaviorally induced plasticity mechanisms are still required in the hippocampus even when the environment is familiar and is consistent with earlier studies that found robust spatial exploration - induced arc mrna expression in dg granular cells even after the environment was experienced for the ninth time . also in agreement with these findings , it was reported by the same group that arc transcription in rats trained in the morris water maze task is similar after overtraining compared to after initial acquisition . all these findings suggest that active spatial exploration induce arc - dependent plasticity mechanisms in the hippocampus every time it is reinstated , regardless of familiarity . strong evidence links ltd , that is , a decrease in the efficiency of synaptic communication , with the formation of object recognition memory , and recent evidence points to a role for arc for this type of recognition memory . for example , exploration of a novel object has been associated with induction of ltd in ca1 network , since novel object exploration during low - frequency stimulation of the schaffer - collateral pathway facilitated ltd in rats [ 31 , 96 ] . recently , it was shown that exposure to a novel environment induced strong dendritic expression of arc mrna in hippocampal ca1 pyramidal neurons , but translation remained tightly repressed . however , further exposure to the same environment lifted the break on arc translation in the dendrite and allowed ampa receptor - dependent ltd to proceed . therefore , an attractive possibility suggested in the same study could be that recognition memory operates in such a way that novelty primes activated neurons for ltd but arc translation remains temporally suppressed , until subsequent experiences with the familiarized stimulus trigger arc translation locally at the dendrite and , therefore , promote long - lasting depression , allowing a sparser memory trace to be established . parallel lines of evidence suggest that a similar mechanism could be involved under different sensorial modalities . seeking to elucidate arc expression dynamics in neocortical networks , a recent work analyzed how a previous exposure to a sound affects arc mrna expression in the auditory cortex after presentation of the same sound on the following day . the same proportion of neurons expressed arc mrna after rats were presented with the sound , whether it was novel or familiar . however , they detected a greater proportion of cells with arc transcript in the cytoplasm specifically after exposure to the familiar sound . these results provide compelling evidence that a single exposure to a noncontingent stimulus could modulate arc expression dynamics in cortical networks and provide further evidence that arc - dependent plasticity mechanisms are still occurring during behavioral familiarity . in our lab we unexpectedly found that familiar saccharin consumption induced higher arc protein accumulation in the insular cortex than novel saccharin , even when the amount of fluid consumed remained constant between the two conditions . strikingly , local infusion of anisomycin in the dorsal hippocampus , a treatment known to affect taste familiarization , prevented the increase of arc protein in the insular cortex observed on the second day . further , immunofluorescence analysis revealed that the greater presence of arc in the familiar condition was due to a dramatic increase in dendritic accumulation of the protein and that the same proportion of cells expressed arc after both novel and familiar taste . the fact that high levels of arc protein expression are still observed even after the execution of a familiarized task could well be explained by memory consolidation - induced epigenetic changes that promote a shift in the transcriptional response of a given circuit upon subsequent reactivations . also , it has been proposed that this sustained arc expression after ongoing experience could serve to maintain the trace in a labile state in order to enable subsequent updating of the memory trace . consistently , extinction of an in vitro classical conditioning task induces similar synaptic levels of arc protein more than initial acquisition does . moreover , recent findings from our lab have shown that de novo arc protein synthesis in the insular cortex upon aversive taste memory retrieval is essential for aversive - to - positive hedonic shift of the taste valence ( guzmn - ramos , venkataraman , morin , and bermdez - rattoni , manuscript in preparation ) . however , the key question is whether arc synthesis is required in asymptotically learned tasks , when no additional information or further updating is involved . experiments from our lab found that optimal dendritic arc protein expression occurred when the taste was presented for the fifth time , that is , when behavioral assessment of taste familiarization ( attenuation of neophobia ) suggests that it is indeed asymptotically familiarized . further , as mentioned earlier , other groups have found that arc protein expression occurs in a similar proportion of cells after exploration of a novel and a familiarized environment . these results indicate that de novo arc protein is required every time a familiarization memory is reactivated , no matter how consolidated it is ; however , no clear loss of function study has addressed this issue . furthermore , most of arc knockdown experiments have used asodns , which only inhibit a fraction of arc translation and are relatively unstable and subject to degradation mitigating their effects . the recent development of more stable asodns , as well as in vivo virus - mediated knockdown experiments , could help address this question with more precision . finally , in addition to plasticity mechanisms induced by memory reactivation , ongoing synaptic modifications must occur offline in order to keep the memory trace stable . in keeping with this , it was discovered that offline wave of arc protein expression in hippocampal networks occurred at 8 h and 24 h after spatial exploration . in the dg , on the other hand , a single 5 min spatial exploration task was shown to produce sustained transcription of arc mrna in granular cells for as long as 8 hours . arc mrna expression in ca1 neurons during rest periods is not random but rather recapitulates previous experiences . importantly , it was shown that the fraction of neurons expressing arc after spatial exploration and expressing it again during a subsequent rest period is highest in ca3 and lowest in the cortex , which is in accordance with the systems consolidation theory . here again , as a possible explanation for these so - called offline genomic reactivations , an epigenetic event , such as dna methylation , could occur during initial acquisition and serve as an indelible footprint that allows for a subsequent round of synapse - specific consolidation to be accomplished every time the same network is solicited . such an epigenetic tag could also alter the rate or susceptibility of transcription of certain genes in an ongoing fashion , in the absence of stimuli . in the future , more extensive characterization of learning - induced epigenetic modifications of arc and other memory - related genes at specific loci will in our view greatly refine our understanding of how memories are dynamically stored and maintained over the range of years . this review examined new findings on the role of arc in long - term synaptic plasticity and memory formation . as we have seen , arc 's unique role in altering network function , possibly as a synaptic contrast enhancer , is reflected by the wide range of brain structures and memory paradigms in which its synthesis is required for ltm formation to proceed . also particularly intriguing are the several models in which its translation is optimally promoted by stimulus familiarity rather than novelty or retrieval of a consolidated memory rather than establishment of a novel one . further information on arc 's regulation mechanisms , particularly at the epigenetic level and on its molecular partners at the synapse should provide helpful insights for the emerging field of the neurobiological basis of memory persistence .

the mainstream view on the neurobiological mechanisms underlying memory formation states that memory traces reside on the network of cells activated during initial acquisition that becomes active again upon retrieval ( reactivation ) . these activation and reactivation processes have been called conjunctive trace . this process implies that singular molecular events must occur during acquisition , strengthening the connection between the implicated cells whose synchronous activity must underlie subsequent reactivations . the strongest experimental support for the conjunctive trace model comes from the study of immediate early genes such as c - fos , zif268 , and activity - regulated cytoskeletal - associated protein . the expressions of these genes are reliably induced by behaviorally relevant neuronal activity and their products often play a central role in long - term memory formation . in this review , we propose that the peculiar characteristics of arc protein , such as its optimal expression after ongoing experience or familiar behavior , together with its versatile and central functions in synaptic plasticity could explain how familiarization and recognition memories are stored and preserved in the mammalian brain .

1. Introduction: Characterization of IEGs and the Particularities of Arc 2. Molecular Mechanisms of Arc-Dependent Synaptic Plasticity 3. The Requirement of Arc for LTM Formation 4. Memory Circuits Reactivations and Ongoing Synaptic Plasticity 5. Familiar/Consolidated Tasks Induce Robust Arc Expression: Abundant Evidence but Still Elusive Function 6. Conclusions and Further Issues

PMC5388047

moderate traumatic physical challenge has been frequently used as a therapeutic modality in traditional chinese medicine , which leads to an anticipation of beneficial health outcome following muscle - damaging exercise . we have previously reported an increased necrosis and white blood cell infiltration in exercised muscle after downhill running , an exercise regimen containing eccentric muscle contraction . however , decreased insulin sensitivity in glucose transport and impaired glycogen storage of skeletal muscle after eccentric muscle contraction has been reported in animals and humans.4 , 5 skeletal muscle , accounted for 40% of body weight , is the major tissue for post - meal glucose uptake . the whole - body insulin sensitivity , assessed by hyperinsulinemic euglycemic clamp technique , suggests that insulin resistance occurs during the first 48 h after eccentric exercise.6 , 7 decreased muscle insulin sensitivity is expected to produce a negative consequence in the whole - body glycemic regulation . based on these findings , we hypothesized that muscle - damaging exercise training will decrease glucose tolerance in men . blood creatine kinase ( ck ) level has been commonly used to measure the levels of exercise - induced muscle damage . histological analysis suggests that blood level of muscle ck should be regarded as a biomarker for post - exercise muscle regeneration.2 , 8 in this study , we assessed oral glucose tolerance together with muscle ck in plasma , two days following the last bout of exercise training containing eccentric muscle contraction . we tested the hypothesis that 1 ) muscle - damaging exercise will decrease the whole - body glucose tolerance ; 2 ) the degree of ck increase is associated with attenuated glucose tolerance in men . twenty - one healthy junior athletes ( 16.3 0.5 years of age ) with no history of musculoskeletal disorders of the lower limbs were enrolled in this study . participants performed barbell front squat and downhill run for 3 consecutive days then allowed to rest for another 2 days ( fig . 1 ) . for ogtt , blood was collected at day 1 before exercise challenge and again at day 4 , 12 h after the last bout of training . to verify the result of muscle damage , blood sample was collected at day 1 ( 24 h ) and day 2 ( 48 h ) for ck analysis after the last exercise bout . participants were instructed to perform 5 sets of barbell front squat for 20 repetitions at 30 pounds a day for 3 consecutive days and allowed to rest for another 3 consecutive days . the 100-meter downhill sprinting consisted of 5 repetitions a day for 3 consecutive days and allowed to rest for another 3 consecutive days . under a 12-h overnight fasted condition , a 500-ml solution containing 75 g of glucose was orally delivered , and blood was taken from the finger at 0 ( before the solution load ) , 30 , 60 and 90 min afterward . blood glucose level was determined by an automated glucose analyzer ( lifescan , inc . , ck ( ckmb isoform ) in plasma is an indicator of muscle regeneration after exercise - induced muscle damage . plasma was obtained by centrifuging the blood at 4 c for 10 min at 3000 rpm and was stored at 80 c until analysis . ck activity was measured using a dt-60 automated hematology analyzer ( kodak co. , rochester , ny , usa ) . student 's t - test was used to compare the mean difference in each variable between the two groups ( low ck group vs. high ck group ) . statistical significance for type i error was set at p < 0.05 for all measures . the current exercise challenge protocol ( fig . 1 ) results in a wide range of muscle ck surge ( table 1 ) . for those participants showing relatively lower ck increases ( fig . 2a ) , glucose levels during ogtt decreased significantly after exercise , indicating an improvement in the glucose tolerance . 2b ) , similar magnitude of improvement on glucose level during ogtt was also reached . data on area under curve ( auc ) in ogtt show no difference in improvement of glycemic control between two groups ( fig . it has been shown that aerobic endurance exercise , as an exercise regimen producing insignificant muscle damage , can improve glucose tolerance . on the other hand , muscle - damaging eccentric muscle contraction results in insulin resistance in glucose transport and glycogen storage of exercised skeletal muscle.4 , 5 based on these early findings , we hypothesized that muscle - damaging exercise will decrease glucose tolerance in humans . our data suggest the benefit of improving glycemic control by a muscle - damaging exercise . therefore , insulin sensitivity of skeletal muscle should significantly influence the whole - body glucose tolerance . the previous report on onset of muscle insulin resistance after eccentric muscle contraction appears to be associated with decreases in glucose transporter glut4 protein in skeletal muscle.4 , 5 , 6 one possibility to explain this discrepancy may be associated with the time after the first bout of exercise challenge . in the current study , our participants conducted 3-day downhill running and measurements were conducted 2 days after the last bout of exercise . glycemic condition may be changing overtime during different stage of the post - exercise recovery . in addition , it has been reported that a single bout of eccentric exercise can result in an increased insulin secretion in response to hyperglycemia . this is based on results from a hyperglycemic clamp assessment , which shows an initial elevation in insulin release at the first 10 min of glucose infusion . our current data show decreased blood glucose during ogtt , suggesting that either insulin sensitivity or increased insulin secretion must take place to lower blood glucose levels . therefore , the result of lower glucose response in ogtt after the muscle damaging exercise is likely associated with concurrent increased response of insulin secretion from pancreatic beta cells and improved insulin sensitivity of exercised muscle . in this study a wide spectrum of muscle damage was produced among different participants . based on our hypothesis , if muscle damage can lower insulin sensitivity , we should observe differences in ogtt response between high ck and low ck group . the most likely explanation is that the exercise challenge damaged relatively weaker muscle fiber with insufficient metabolic function . it is quite easy to perceive that healthy muscle fibers have greater chance of survival after an entropic challenge . there may be a great variation in the proportion of unhealthy versus healthy muscle fibers among participants due to individual 's variation in training status or lifestyle . thus the exercise challenge lowers the unhealthy population of muscle fibers of all participants to a similar level , regardless their pre - existing percentage of metabolically weaker muscle fibers . human body may be considered as a society composed by cells , where the principle of darwinian natural selection should apply . it has been shown that all - cause mortality decreases as exercise intensity increases,11 , 12 suggesting that removing relatively less robust cells in human body by greater challenge can bring more beneficial outcome . we speculate that enhance muscle fiber renewal may be the most important mechanism accounted for the beneficial outcome of the entropic challenge in glycemic control . skeletal muscle cells in human body are relatively short - lived compared to brain and is continuously dying and regenerating overtime . during early stage of muscle development , glut1 protein ( low km transporter ) is predominantly expressed followed by increasing glut4 expression as muscle mature . therefore , the knowledge generalized from the study for older population deserves more experimental confirmation . it is generally known that the individuals having greater needs for better glycemic control are those middle - aged or elderly people . therefore , the current result does not provide direct answer with regard to whether muscle - damaging exercise is able to improve glucose tolerance at higher age level . in conclusion , according to previous finding on decreased insulin sensitivity of skeletal muscle after eccentric muscle damaging exercise , we hypothesized that downhill running with resistance exercise will result in deterioration in ogtt . this finding implicates that metabolically weaker muscle fibers are replaced by newly generated cell population after muscle - damaging eccentric exercise .

tissue damage is regarded as an unwanted medical condition to be avoided . however , introducing tolerable tissue damages has been used as a therapeutic intervention in traditional and complementary medicine to cure discomfort and illness . eccentric exercise is known to cause significant necrosis and insulin resistance of skeletal muscle . the purpose of this study was to determine the magnitude of muscle damage and blood glucose responses during an oral glucose tolerance test ( ogtt ) after eccentric training in 21 young participants . they were challenged by 5 times of 100-meter downhill sprinting and 20 times of squats training at 30 pounds weight load for 3 days , which resulted in a wide spectrum of muscle creatine kinase ( ck ) surges in plasma , 48 h after the last bout of exercise . participants were then divided into two groups according the magnitude of ck increases ( low ck : + 48% 0.3 ; high ck : + 137% 0.5 , p < 0.05 ) . both groups show comparable decreases in blood glucose levels in ogtt , suggesting that this muscle - damaging exercise does not appear to decrease but rather improve glycemic control in men . conclusion : the result of the study rejects the hypothesis that eccentric exercise decreases glucose tolerance . improved glucose tolerance with ck increase implicates a beneficial effect of replacing metabolically weaker muscle fibers by eccentric exercise in darwinian natural selection fashion .

Introduction Methods Results Discussion Conflict of interest

PMC4992994

a family member s chronic illness and its economic and psychosocial consequences involve the entire family and affect their lifestyle . studies show that family caregivers of patients with a chronic illness experience a vast range of physical and emotional distresses and psychological symptoms , including depression , anxiety , anger , despair , and feelings of guilt and shame ( 1 , 2 ) . chronic renal failure ( crf ) leads to significant changes in the lives of patients and their families . the need for frequent hemodialysis and its associated health problems reduce patients energy and negatively affect their ability to work and to perform routine daily activities ( 3 - 5 ) . about 2 to 3 percent of people worldwide and more than 10% of americans are affected by crf ( 6 ) . the disease is increasing in developing countries ; its prevalence in iran increased from 238 cases per million people in 2000 to 354 cases per million in 2006 ( 7 ) . a few treatment options are available for patients with crf , including hemodialysis , peritoneal dialysis , and kidney transplantation . in iran , 47.7% of all patients with crf use hemodialysis ; and a total of 25,934 patients were under chronic hemodialysis in 2013 ( 6 - 8 ) . although the widespread availability of hemodialysis saves and prolongs the lives of thousands of patients with end - stage renal diseases , these patients suffer from many problems and complications ( 9 , 10 ) . hemodialysis patients experience a high degree of disability , loss of functions , and dependency on their caregivers , particularly family caregivers ( 11 , 12 ) . studies showed that family caregivers have an exclusive role in caring for these patients and therefore are under considerable physical and psychological pressure ( 1 , 13 - 15 ) . in a recent study , abbasi et al . investigated the burden on caregivers of hemodialysis patients and reported that 74.2% of them experienced severe burden ( 16 ) . over time , as patients worsen , their caregivers burden also increases and they experience more physical and psychological problems , social isolation , and disruption in family relationships , which might finally lead to shortcomings or discontinuing the patient care ( 1 , 17 ) . therefore , these family caregivers are at risk of disease and are sometimes referred to as hidden patients ( 18 ) . in hemodialysis centers , health professionals are responsible for patient care . however , at home , the patients relatives undertake this role . these caregivers are often deficient in knowledge and skills related to patient care and lack social support or support from the healthcare system . with disease progression , patients became more disabled and caregivers are confronted with more complex caring needs ( 19 - 21 ) . with an increase in patients caring needs , the burden on caregivers increases . then , their quality of life is reduced ( 12 ) and they experience more anger , anxiety , and the inability to cope with their caring roles ( 22 - 24 ) . however , few interventional studies are available on the alleviation of the burden on caregivers of hemodialysis patients . in a study in turkey , mollaoglu et al . investigated the effects of education related to home care in patients undergoing hemodialysis on caregiver burden and reported that education was effective in reducing caregivers ; burden ( 20 ) . in another study , khorami markani et al . examined the effect of a family - centered educational program on the home care knowledge of the caregivers of patients under chronic hemodialysis and reported positive effects ( 25 ) . in addition to knowledge on caring , caregivers need appropriate skills in communicating with patients and dealing with stressful situations ( 12 , 26 ) to cope with such a stressful and demanding life . there are two categories of coping strategies : problem - focused strategies ( based on one 's ability to manage the environmental event ) and emotion - focused strategies ( that focus on changing the emotions caused by a stressful situation ) ( 27 , 28 ) . a number of studies have investigated the effectiveness of coping strategies on caregiver burden among caregivers of patients with dementia , mental disorders , heart failure , and cancer ( 23 , 24 , 29 , 30 ) and demonstrated positive effects . however , the caregivers of hemodialysis patients have largely been neglected ( 22 , 31 ) . although some descriptive and review studies are available and have reported moderate to severe levels of burden on these caregivers ( 14 , 16 , 31 ) , only one interventional study is available on this group , and this study has no control group ( 20 ) . no additional studies are available on the effectiveness of coping strategies on the burden of care among these caregivers . this study aimed to examine the effectiveness of problem - focused coping strategies ( communication skills , anger management , and deep breathing ) on the burden on caregivers of hemodialysis patients . a randomized controlled clinical trial was conducted on caregivers of hemodialysis patients referred to shahid hasheminejad hemodialysis center in tehran , iran . inclusion criteria for the caregivers were as follows : being a patient s first - degree relative , having the responsibility for the home care of his or her hemodialysis patient , willingness to participate in the study , at least 18 years of age , writing and reading literacy , having no known psychological or neurological disorders , having no severe family conflict , and not being a healthcare worker . inclusion criteria for the patients were as follows : performing regular hemodialysis for at least two months , at least three times a week , and for 3 - 4 hours in each session ; having no history of kidney transplantation ; and having a family caregiver to do home care . the lack of appropriate cooperation by the caregiver , participation in similar training courses , the occurrence of a family crisis ( i.e. , divorce , financial crisis , the death of a first - degree family member ) during the study , a subject s decision to withdraw from the study , the absence of even a training session , and booking the patient on the kidney transplantation list were selected as exclusion criteria . due to the lack of similar studies on caregivers of hemodialysis patients , the sample size was estimated based on a pilot study on 16 caregivers that were equally placed into two groups of eight . after administering the caregiver s burden inventory ( cbi ) , four sessions similar to the main study were conducted in one group , and after one month , the cbi was again administered to both pilot groups . the mean burden in the intervention group was changed from 83.51 12.47 to 63.21 14.28 while it did not significantly change in the control group ( 84.64 13.21.vs . then , using the following parameters ( = 0.10 , = 0.01 , 1 = 81.01 , and 2 = 63.21 , s1 = 12.67 , s2 = 14.28 ) , 18 subjects were estimated to be needed in each group . however , considering the possible dropouts and for more confidence , we doubled the sample size and recruited 38 subjects in each group . the first part included a demographic questionnaire including questions on the caregiver s and the patient s demographic data , such as the caregiver s age , gender , marital status , education level , job , type of family relationship with the patient , financial status , having a known physical illness , and the size of their family as well as the duration of the patient s disease , duration of using regular hemodialysis , history of kidney transplantation , dialysis association membership , having active insurance coverage , and the type of insurance coverage . in addition , there was a question on a four - point likert scale on the patient s ability to perform his or her own personal tasks ( very low , low , high , and very high ) . the farsi version of the cbi was used as the second part of the study instrument . the cbi is composed of 24 items in five subscales , including time - dependence burden ( items 1 to 5 ) , developmental burden ( items 6 to 10 ) , physical burden ( items 11 to 14 ) , social burden ( items 15 to 19 ) , and emotional burden ( items 20 to 24 ) . all items are responded on a five - point likert scale ranging from 1 (= never ) to 5 (= nearly always ) . moreover , the scores between 24 and 39 , 40 and 71 , and 72 and 120 are categorized as low , moderate , or severe burden , respectively ( 32 , 33 ) . this scale was translated to farsi by abbasi et al . and its validity and reliability were confirmed through content validity and internal consistency ( cronbach 's alpha = 0.90 ) ( 16 ) . after approval of the study was obtained , the first researcher referred to the aforementioned hemodialysis center , and through a file review and interviews with the patients , those with inclusion criteria were found . then , the researcher made telephone contact with the patients main caregivers and , through telephone interviews , assessed their eligibility , informed them that a study is starting to investigate caregiver s burden , and invited them to participate in the study . caregivers who agreed to take part were informed that they would be involved the study for about two months and would be asked to complete the questionnaires two times during the study . then , all of them were invited to attend a session in the hall of the dialysis center to complete the study instrument and were informed that after a while they would be invited to attend several educational sessions . subsequently , through a coin - tossing method , caregivers of the patients who referred on even or odd days of the week were randomly assigned to either the intervention group or the control group , respectively . then , the 38 caregivers in the intervention group were allocated into five small subgroups of five to eight , and each subgroup participated in four training sessions on problem - focused coping strategies ( i.e. , proper communication , anger management , and deep breathing ) that were held twice a week , in two consecutive weeks , and each session lasted for about an hour . all training sessions were delivered by an expert psychiatric nurse who was previously trained and tested to facilitate group discussions . each session consisted of a combination of a short powerpoint facilitated lecture , a group discussion , a question and answer period , and a role playing . at the end of the first session , an educational booklet related to the issue the content validity of the educational booklet was confirmed by 10 nursing professors in the tehran , iran and shahid - beheshti universities of medical sciences . six weeks after the last educational session , all subjects in the experimental group and the control group were again invited to attend a session in the hall of the dialysis center and responded to the study instrument . this study was approved by the ethics committee of iran university of medical sciences ( grant no . permission was also sought from the authorities in the university and the shahid hasheminejad dialysis center . all participants were briefed about the study s purposes and the voluntary nature of their participation . they all signed a written informed consent , were assured of the anonymity and confidentiality of the data , and were also reminded that they can withdraw from the study at any time . the researchers were sensitive to preserving the participants rights according to the helsinki ethical declaration . to observe ethics , the caregivers in the control group also received the educational booklet after the last assessment . descriptive statistics such as frequencies , percentage , mean , and standard deviation were calculated . the kolmogorov - smirnov test was used to examine the normal distribution of quantitative variables . the chi - square test was used to compare the nominal and categorical variables , such as gender , marital status , education level , job , and having a chronic co - morbidity , between the two groups . fisher s exact test was used to compare the two groups in terms of the patients duration of hemodialysis , type of insurance coverage , dialysis association membership , and caregivers relationship with the patient and financial status . the independent - samples t - test was used to compare the caregivers mean age and mean burden scores in the two groups . the mann - whitney u test was also used to compare the patients in the two groups in terms of their ability to perform their own personal tasks . the first part included a demographic questionnaire including questions on the caregiver s and the patient s demographic data , such as the caregiver s age , gender , marital status , education level , job , type of family relationship with the patient , financial status , having a known physical illness , and the size of their family as well as the duration of the patient s disease , duration of using regular hemodialysis , history of kidney transplantation , dialysis association membership , having active insurance coverage , and the type of insurance coverage . in addition , there was a question on a four - point likert scale on the patient s ability to perform his or her own personal tasks ( very low , low , high , and very high ) . the farsi version of the cbi was used as the second part of the study instrument . the cbi is composed of 24 items in five subscales , including time - dependence burden ( items 1 to 5 ) , developmental burden ( items 6 to 10 ) , physical burden ( items 11 to 14 ) , social burden ( items 15 to 19 ) , and emotional burden ( items 20 to 24 ) . all items are responded on a five - point likert scale ranging from 1 (= never ) to 5 (= nearly always ) . moreover , the scores between 24 and 39 , 40 and 71 , and 72 and 120 are categorized as low , moderate , or severe burden , respectively ( 32 , 33 ) . its validity and reliability were confirmed through content validity and internal consistency ( cronbach 's alpha = 0.90 ) ( 16 ) . after approval of the study was obtained , the first researcher referred to the aforementioned hemodialysis center , and through a file review and interviews with the patients , those with inclusion criteria were found . then , the researcher made telephone contact with the patients main caregivers and , through telephone interviews , assessed their eligibility , informed them that a study is starting to investigate caregiver s burden , and invited them to participate in the study . caregivers who agreed to take part were informed that they would be involved the study for about two months and would be asked to complete the questionnaires two times during the study . then , all of them were invited to attend a session in the hall of the dialysis center to complete the study instrument and were informed that after a while they would be invited to attend several educational sessions . subsequently , through a coin - tossing method , caregivers of the patients who referred on even or odd days of the week were randomly assigned to either the intervention group or the control group , respectively . then , the 38 caregivers in the intervention group were allocated into five small subgroups of five to eight , and each subgroup participated in four training sessions on problem - focused coping strategies ( i.e. , proper communication , anger management , and deep breathing ) that were held twice a week , in two consecutive weeks , and each session lasted for about an hour . all training sessions were delivered by an expert psychiatric nurse who was previously trained and tested to facilitate group discussions . each session consisted of a combination of a short powerpoint facilitated lecture , a group discussion , a question and answer period , and a role playing . at the end of the first session , an educational booklet related to the issue the content validity of the educational booklet was confirmed by 10 nursing professors in the tehran , iran and shahid - beheshti universities of medical sciences . six weeks after the last educational session , all subjects in the experimental group and the control group were again invited to attend a session in the hall of the dialysis center and responded to the study instrument . this study was approved by the ethics committee of iran university of medical sciences ( grant no . permission was also sought from the authorities in the university and the shahid hasheminejad dialysis center . all participants were briefed about the study s purposes and the voluntary nature of their participation . they all signed a written informed consent , were assured of the anonymity and confidentiality of the data , and were also reminded that they can withdraw from the study at any time . the researchers were sensitive to preserving the participants rights according to the helsinki ethical declaration . to observe ethics , the caregivers in the control group also received the educational booklet after the last assessment . descriptive statistics such as frequencies , percentage , mean , and standard deviation were calculated . the kolmogorov - smirnov test was used to examine the normal distribution of quantitative variables . the chi - square test was used to compare the nominal and categorical variables , such as gender , marital status , education level , job , and having a chronic co - morbidity , between the two groups . fisher s exact test was used to compare the two groups in terms of the patients duration of hemodialysis , type of insurance coverage , dialysis association membership , and caregivers relationship with the patient and financial status . the independent - samples t - test was used to compare the caregivers mean age and mean burden scores in the two groups . the mann - whitney u test was also used to compare the patients in the two groups in terms of their ability to perform their own personal tasks . the majority of caregivers ( 77.6% ) had no physical disorder ; most of their patients had a low or very low ability to perform their own personal tasks ( 79% ) and were using regular hemodialysis for more than two years ( 76.35% ) , and all had insurance coverage ( table 2 ) . no significant difference was found between the two groups baseline mean caregivers burden scores before the intervention ( p = 0.308 ) . however , the mean caregivers burden in the intervention group was reduced , and the two groups were significantly different at the end of the study ( p < 0.001 ) ( table 3 ) . overall , 0% , 15.8% , and 84.2% of the caregivers in the control group and 0% , 10.5% , and 89.5% of caregivers in the intervention group experienced low , moderate , or severe burden before the intervention ( p = 0.497 ) . however , at the end of the study , the rates of low , moderate , or severe burden in the intervention group were reduced to 0% , 94.7% , and 5.3% , whereas these rates did not significantly change in the control group ( p < 0.001 ) . the two groups mean scores on caregivers burden were also compared in terms of their demographic variables , and no significant differences were found ( table 4 ) . values are expressed as mean sd . the mean scores of the different domains of caregivers burden were also compared between the two groups . before the intervention , no significant difference was observed between the two groups except in the domain of emotional burden . however , the mean scores of the intervention group decreased in all domains after the intervention , and all domains were different in the two groups at the end of the study ( p < 0.001 ) ( table 5 ) . abbreviation : ci , confidence interval . this study is the first to evaluate the effectiveness of a training program involving problem - focused coping strategies on the burden on caregivers of hemodialysis patients . the present study showed that learning coping strategies can reduce caregivers burden of care . this finding , along with previous studies , shows the importance of training in supporting caregivers of patients with chronic disorders , such as users of regular hemodialysis ( 13 , 31 , 34 ) . family caregivers should not only take care of themselves , but also simultaneously meet the patients caring needs . consequently , they experience high levels of physical , emotional , financial , and social burden , which change their lifestyle ( 14 ) . at the start of the current study , more than 80% of the family caregivers demonstrated symptoms of severe burden . this finding is consistent with some of the previous studies that investigated the burden on caregivers of hemodialysis patients ( 14 - 16 , 20 ) . caregivers of hemodialysis patients experienced low levels of burden ( 35 ) , which might be attributed to the patients and caregivers characteristics , such as higher levels of caregivers education , higher levels of patients self - caring abilities , and performing hemodialysis at night . a number of the earlier studies investigated the relationship between caregivers burden and certain demographic variables . for instance , abbasi et al . showed a direct relationship between caregivers burden and income ( 16 ) . ( 20 ) also reported an association between caregivers burden and education , indicating caregivers with higher education levels experience lower levels of burden . however , in the current study , we did not find any significant relationship between caregivers burden and demographic variables . at the beginning of this study , no significant difference was observed between the two groups in different domain of caregivers burden except for emotional burden , which was higher in the intervention group . moreover , both groups expressed the highest levels of burden in the domains of developmental , physical , and time dependence . caregivers experienced higher levels of burden in the emotional , social , and developmental domains ( 16 ) . these inconsistencies might be attributed to the age of the caregivers , which was higher on average in the present study . previous studies have also shown that caregivers burden in the physical , developmental , and time - dependence domains increases with age , whereas the burden in the emotional domain decreases . most of the caregivers in this study were females who were daughters or wives of the hemodialysis patients . evidence shows that most caregivers of chronic patients in asian families are females ( 5 ) . have also reported that female caregivers who are family members of patients are usually more sentimental and sensitive to patients caring needs and also have a greater ability than men to manage problems and establish intimate relationships with patients ( 20 ) . the caregiving role can be associated with feelings of compassion , love , and intimacy in relationships . however , an increases in the patients and caregivers caring needs along with the caregivers lack knowledge and skills related to coping strategies can increase their burden ( 16 , 22 ) . studies have shown that family caregivers burden can not only increase their physical complaints , but also may lead to feelings of guilt , disappointed , loneliness , depression , anger , stress , and a lack of freedom , which may result in severe psychological problems ( 2 , 35 ) . the present study , along with several previous investigations , confirmed that family caregivers are often lacking appropriate coping strategies and need to be supported . several previous studies on the caregivers of patients with cancer , diabetes , and cardiac and mental disorders have shown that training the caregivers on coping strategies can significantly reduce their burden and increase their self - esteem , perceived health , and quality of life and eventually increases the quality of patient care ( 12 , 23 , 24 , 26 , 27 , 30 , 37 ) . confirming the intervention used in the present study , a previous study on family caregivers of hemodialysis patients revealed that those who often use problem - focused coping strategies experience fewer burdens than those who use emotion - focused coping strategies ( 38 ) . furthermore , etemadifar et al . ( 37 ) reported that supportive educative group interventions and group psycho - educational programs , such as the program implemented in the current study , are more effective in reducing caregivers burden , enhancing their own perceived health , and improving patient care . it seems that learning problem - focused coping strategies helps caregivers gain more control , not only over stressful situations , but also over their own behaviors and time management , allowing them to feel less burdened in all aspects of their lives , particularly in the developmental , emotional , social , and time - dependence domains . as the present study showed , the beneficial effects of learning problem - focused continues for weeks or perhaps for months , and the longevity of the effects might be augmented through some reinforcement strategies , such as intermittent repetition of the program . studies have shown that besides knowledge and skills on coping strategies , caregivers of hemodialysis patients need counseling , empathy , and psychological support to cope with their caregiving roles ( 31 , 34 ) . isenberg et al . ( 19 ) and khanjari et al . ( 27 ) showed that group discussions and sharing experiences among caregivers are effective in providing ways to give and receive empathy and psychological support . confirming the findings of previous studies , the present study also showed that these strategies , along with educating the caregivers on problem - focused coping strategies , were significantly effective in reducing the burden on caregivers of hemodialysis patients . this study was conducted only on caregivers of patients in a dialysis center ; the small sample size and the relatively short follow - up period can be considered limitations to generalize the findings of this study . therefore , the replication of similar studies with larger sample sizes and longer periods of follow - up is recommended . moreover , as in any questionnaire study , the caregivers responses to the questionnaire might have been affected by their psychological condition , and this was not under the full control of the researchers . in conclusion , the current study showed the effectiveness of problem - focused coping strategies on reducing the burden on caregivers of hemodialysis patients . presently , there are no ongoing programs focused on educating family caregivers in the healthcare system of iran , and caregivers of hemodialysis patients are completely ignored . authorities and policymakers in the healthcare system are responsible for developing strategies to integrate educational programs , such as the program implemented in the current study , into the country s healthcare system . moreover , educating the family caregivers should be integrated into all patient education programs running for all chronic patients , including chronic renal failure and hemodialysis patients . the importance of educating and empowering family caregivers should also be emphasized in in - service and continuing nursing education . particular courses of training family caregivers with a special focus on problem - focused coping strategies is also recommended to be integrated into the curriculum of nursing , both at the undergraduate and postgraduate levels .

backgroundstudies have shown that family caregivers of hemodialysis patients experience high levels of burden . however , these caregivers are often neglected , and no studies are available on the effectiveness of coping strategies on the burden of care among these caregivers.objectivesthis study aimed to examine the effectiveness of problem - focused coping strategies ( communication skills , anger management , and deep breathing ) on the burden on caregivers of hemodialysis patients.patients and methodsa randomized controlled clinical trial was conducted on 76 family caregivers of hemodialysis patients referred to shahid hasheminejad hemodialysis center in tehran , iran . the subjects were equally allocated into two groups of 38 . through a coin - tossing method , caregivers of patients who referred on even or odd days of the week were randomly assigned into the intervention group or the control group , respectively . the intervention group received four training sessions on problem - focused coping strategies , but the control group did not receive any intervention . both groups answered the caregiver s burnout inventory at the start and six weeks after the last educational session . descriptive statistics , chi - square , fisher s exact test , independent - samples t - test , and mann - whitney u test were used to analyze the data.resultsthe majority of caregivers ( 54% ) were in the age range of 35 - 55 years , female ( 68.4% ) , and married ( 70% ) . no significant difference was found between the baseline mean caregivers burden scores of the intervention and control groups ( 88.56 11.74 vs. 84.97 15.13 , p = 0.308 ) . however , the mean caregivers burden in the intervention group decreased , and the two groups were significantly different at the end of the study ( 58.77 6.64 vs. 87.84 11.74 , p < 0.001).conclusionsthe current study showed the effectiveness of problem - focused coping strategies on reducing the burden on caregivers of hemodialysis patients . authorities and policymakers in the healthcare system are responsible for developing strategies to integrate educational programs , such as the program implemented in the current study , into the country s healthcare system .

1. Background 2. Objectives 3. Patients and Methods 3.1. The Study Instrument 3.2. Intervention 3.3. Ethical Considerations 3.4. Data Analysis 4. Results 5. Discussion

PMC4120049

at the end of their pgy-2 year in june 2012 , 12 prospective chief residents underwent a half - day training session using a curriculum customized by program leadership called lead ( leadership , education , advocacy and development ) . the training included several topics presented by two former full - year pgy-3 chief residents serving in administrative roles , as well as residency program leaders including the chairperson , program directors , and core faculty . session themes included communication , motivation , delegation , feedback , team - building , task completion , clinical teaching , running meetings and career success . in addition , based on self - identified interest or faculty evaluation , some residents were assigned additional roles as directors for the following boards : conference curriculum , continuity clinic , recruitment , community advocacy / esprit de corp , and transition to residency . each of these groups worked closely with faculty members to design strategies for the upcoming year . additionally , each chief met with core faculty early in the month during which they were chief ( an elective or outpatient clinic rotation , in most cases ) to review the chief resident curriculum . each third - year chief received a $ 500 stipend ( one - twelfth of the annual department budget for chief residents ) . after the rotating chief and leadership curriculum was implemented , survey questionnaires were sent to all 29 internal medicine residents in the program at the end of each month to evaluate each chief resident 's performance . these questionnaires were adapted from published literature using a previously evaluated reference model ( 1 , 5 ) ; respondents utilized a 010 scale for each question . the rotating chief for any given month was excluded from participating in the group surveys ; however , he or she was asked to participate in a separate set of surveys that evaluated his or her leadership capacities as demonstrated throughout the chief rotation . all other participating residents evaluated the rotating chief on his or her performance throughout the rotation . thirteen valid junior resident responses were obtained for the chief resident evaluation survey : eight from pgy-1 and five from pgy-2 . the data from the pgy-1 respondents yielded similar means to that of the pgy-2 class , so the two classes were merged for final analysis . junior residents reported that the chief residents elicited input from others ( 6.8 ) , were committed to the team ( 6.8 ) , resolved conflict ( 6.7 ) , ensured efficiency , organization and productivity of the team ( 6.7 ) , participated actively ( 7.0 ) , and managed resources ( 6.6 ) . pgy-3 ( n=16 ) responses provided higher averages on all the above measures ( table 1 ) . the pgy-3 class ( n=9 ) also gave higher ratings of the chiefs teaching skills than did the junior residents ( n=7 ) . rating of chief residents by postgraduate year of rater self - evaluation by the chief residents ( n=5 ) revealed that they rated their ability in leadership low ( mean 6.0 , sd 1.2 ) ; they also reported low comfort with being chief resident ( mean 5.8 , sd 0.8 ) . they were more comfortable running a morning report ( mean 8.4 , sd 1.5 ) or noon conference ( mean 7.6 , sd 1.5 ) than a quality improvement study ( mean 6.8 , sd 1.1 ) . additionally , pgy-1 and -2 residents appeared to be more critical of the leadership and teaching skills of the third - year chief residents than were their own pgy-3 cohorts . first , the responses from the pgy-3 class were self - ratings and may have inherent bias . second , the study was performed at a single internal medicine program , and may thus have limited generalizability to other residency programs . hypothesis testing was not applied to this data due to a lack of power to detect a statistically significant difference . despite these limitations , we suggest that there may be a role for introducing a rotating pgy-3 chief curriculum in residency programs that do not have pgy-4 chief residents . it may be worthwhile to explore on a larger scale whether obtaining input from junior residents would correlate with the success of elected chief residents . interestingly , 86% of family medicine programs utilize input from residents in the selection of chief residents ( 7 ) . it may be useful to extend the study to include internal medicine residency programs that have both pgy-4 and pgy-3 chiefs , and to gather data on the selection processes of chiefs as well as quantitative ratings of the chiefs productivity , leadership , resident satisfaction , and other metrics that would help other programs determine how to improve selection of , and role definition for , chief residents . the feasibility of accomplishing this through a rotating pgy-3 chief residency curriculum was explored at one small program , and larger studies are indicated .

introductionthe role of the internal medicine chief resident includes various administrative , academic , social , and educational responsibilities , fulfillment of which prepares residents for further leadership tasks . however , the chief resident position has historically only been held by a few residents . as fourth - year chief residents are becoming less common , we considered a new model for rotating third - year residents as the chief resident.methodsonline surveys were given to all 29 internal medicine residents in a single university - based program after implementation of a leadership curriculum and specific job description for the third - year chief resident . chief residents evaluated themselves on various aspects of leadership . participation was voluntary . descriptive statistics were generated using spss version 21.resultsthirteen junior ( first- or second - year ) resident responses reported that the chief residents elicited input from others ( mean rating 6.8 ) , were committed to the team ( 6.8 ) , resolved conflict ( 6.7 ) , ensured efficiency , organization and productivity of the team ( 6.7 ) , participated actively ( 7.0 ) , and managed resources ( 6.6 ) . responses from senior residents averaged 1 point higher for each item ; this pattern repeated itself in teaching evaluations . chief resident self - evaluators were more comfortable running a morning report ( 8.4 ) than with being chief resident ( 5.8).conclusionthe feasibility of preparing internal medicine residents for leadership roles through a rotating pgy-3 ( postgraduate year ) chief residency curriculum was explored at a small internal medicine residency , and we suggest extending the study to include other programs .

Methods Results Discussion Conclusion Conflict of interest and funding

PMC4304055

similar to fingerprints , the pattern of wrinkles on the lips also has individual characteristics . numerous elevations and depressions that form a characteristic pattern on the external surface of lip are referred to as lip prints . lip prints are easily obtained at the crime scene from various materials such as clothing , cups , glasses , cigarettes , doors , etc . they also possess furrows that can be classified into various types for identification of an individual . in 1902 , r. fischer , an anthropologist , first described the biological phenomenon of systems of furrows on the red part of human lip . use of lip prints in personal identification and criminalization was first recommended by edmond locard . he proved in an investigation that the characteristics of lips formed by lip grooves are as individually distinctive as the ridge characteristics of finger prints . until 1950 , however , anthropology merely mentioned the existence of the furrows on the lip prints . two japanese scientists , y. tsuchihashi and t. suzuki ( 19681971 ) , carried out a research on 1364 persons at the department of forensic odontology at tokyo university . it was established that the arrangement of lines on the red part of human lips is individual and unique for each human being . the lip prints are classified as follows : type i- a clear - cut groove running vertically across the lip type i- partial - length groove of type i type ii- a branched groove type iii- an intersected groove type iv- a reticular pattern type v- the grooves do not fall into any of the types i iv and can not be differentiated morphologically . types i , i , and ii are predominant in females and types iii , iv , and v in males . the lip prints are classified as follows : type i- a clear - cut groove running vertically across the lip type i- partial - length groove of type i type ii- a branched groove type iii- an intersected groove type iv- a reticular pattern type v- the grooves do not fall into any of the types i iv and can not be differentiated morphologically . types i , i , and ii are predominant in females and types iii , iv , and v in males . the study population consisted of 200 first to final year bds students of k. d. dental college and hospital , mathura , uttar pradesh , india . all the subjects ( females = 100 , males = 100 ) belonged to the age group of 1726 years . ethical clearance for conducting the study was obtained from the ethical committee of k. d. dental college prior to the start of the study . before data collection , the purpose and procedure of the study were thoroughly explained to the subjects . also , a written informed consent was obtained from each subject . those students who were present at the time of study and who were free from any lesions on their lips were included in the study . subjects with any lip anomalies or any lesions on the lips , those with any known allergy to lip stick , and those who were not willing to participate were excluded from study . dark - colored , non - glossy , less moisture containing lip stick was used to obtain the lip prints of the subjects . after cleaning the surface of the lip , the lip stick was allowed to settle for a minute , after which a transparent cellophane tape slightly larger than the thickness of the lip was uniformly and gently applied to the lip from right to left . all the lip prints were collected by one assistant who coded all the prints and kept in record the name and sex of the respective individual . however , the sex of lip prints was not disclosed to the examiner , so as to reduce the errors while interpreting the results of the study . the pattern of lip prints was studied quadrant wise [ figure 1 ] by using a magnifying lens by one examiner . patterns of lip prints were classified according to the classification given by suzuki and tsuchihashi . the results obtained were later on verified from the coded data collected by the assistant . lip print division quadrant wise the length and thickness of the lips were measured using standard sliding calipers . length of the lip was measured between the outermost visible points on the lip print at the angle of the mouth . thickness of the lip was measured for the upper and lower lips in the midline . chi - square test was used to analyze the lip print patterns in this study . in our study , 78% male lip prints and 84% female lip prints were diagnosed correctly by the examiner [ figure 2 ] . overall , 162 ( 81% ) were diagnosed correctly and 38 ( 19% ) were diagnosed wrong [ figure 3 ] . accuracy of gender determination by the examiner overall accuracy of cheiloscopy in gender determination the most common lip patterns found in males were type iii and type iv ( 20% each ) , followed by type v ( 18% ) , type i ( 12% ) , type i , type ii , and mixed type ( 10% each ) , as compared to females where the commonest patterns were type i and type i ( 25% each ) , followed by type ii ( 17% ) , type iii and type iv ( 10% ) , mixed type ( 8% ) , and type v ( 5% ) . when the lip print patterns were compared , a statistically significant difference was found between males and females ( p 0.05 ) [ table 1 ] . distribution of lip print pattern types in both males and females the most common types of lip prints observed in males were as follows : quadrant a , type iii ( 8% ) ; quadrant b , type iv ( 5% ) ; quadrant c , type iv ( 5% ) ; and quadrant d , type iii and type v ( 5% each ) [ table 2 ] . quadrant wise distribution of different types of lip prints in males the most common types of lip prints observed in females were as follows : quadrant a , type i ( 8% ) ; quadrant b , type i ( 8% ) ; quadrant c , type i ( 7% ) ; and quadrant d , type i , type i , and type ii ( 4% each ) [ table 3 ] . quadrant wise distribution of different types of lip prints in females regarding the thickness of lip prints in males , the average thickness of the upper lip was 13.3 mm as compared to 9.5 mm in females ; the average difference between males and females was 3.8 mm for the upper lip . the maximum and minimum thicknesses of the upper lip in males were found to be 18.9 mm and 12.5 mm , respectively . in females , the maximum and minimum thicknesses of the upper lip the average thickness of the lower lip in males was 14.0 mm as compared to 11.1 mm in females , and the average difference between males and females was 2.9 mm for the lower lip . the maximum and minimum thicknesses of the lower lip in males were 16.9 mm and 11.5 mm , respectively . in females , the maximum and minimum thicknesses of the lower lip were 15.4 mm and 6.9 mm , respectively . cheiloscopy has been proven to be a reliable technique to establish the correct identity of a person . absence of population - specific and gender - specific data can hamper its application as evidence . hence , analysis and testing of such data in various population groups has become absolutely essential . in our study , 78% males and 84% females were diagnosed correctly by the examiner , in comparison to a study done by dongarwar et al . overall , in our study , out of 200 participants , 81% participants were diagnosed correctly , compared to the studies of bajpai et al . and sharma et al . this proves that cheiloscopy is a unique method for identification of sex . in the present study , the most common lip print patterns found in males were types iii and iv ( 20% each ) . in which type i pattern was the most common among males and in the study of malik et al . however , the studies conducted by arif et al . , sheetal et al . , and kautilya et al . showed that the most common patterns found in males were types iv , v , and iii , respectively . however , the most common lip patterns found in females examined in the present study were type i and type i ( 25% each ) . similarly , malik and goel also reported that type i and type i were the most common patterns in females , while in the study done by kautilya et al . , the most common pattern found was type i ( 30% ) . in the study conducted by kapoor and tiwari , type iii pattern was most frequently present in both males and females . a statistically significant difference ( p 0.05 ) in lip print patterns between males and females was found in the study done by xu et al . and gondivakar et al . , which is in accordance with our study . in our study , type iii was the most common in quadrant a among males and types i and i were the most common in quadrants a , b , and c among females . in the study done by amith et al . , type i was the most predominant pattern in the first and second quadrants . however , in the study conducted by verghese et al . , type iv pattern was found to be the most predominant pattern in the middle portion of the upper lip . the present study revealed the average thickness of the upper lip was 13.3 mm as compared to 9.5 mm in females . the average thickness of the lower lip in males was 14.0 mm as compared to 11.1 mm in females . a difference of about 3.8 mm in the lip thickness of upper lip and about 2.9 mm in the lip thickness of the lower lips between males and females were seen . male lips were found to be thicker as compared to female lips . also , in the study conducted by kautilya et al . male lip prints were thicker than female lip prints and this finding could be helpful to differentiate the sexes . limitations of the study were that since the lip print was produced with a substantially mobile portion of the lip , the same person can produce different lip prints according to the pressure , direction , and method used in taking the print . the existence of some pathological conditions ( lymphangiomas , congenital lip fistula , syphilis , lip cheilitis , etc . ) can invalidate the cheiloscopic study . using the lip print pattern for identification of a person is one of the unique practices . male lips were found to be thicker as compared to female lips in our study . therefore , lip print pattern can be used as one of the weapons for personal identification of an individual .

aim : to determine the sex of an individual from the configuration of lip prints.materials and methods : this study was conducted on 200 first to final year bds students of k. d. dental college and hospital , mathura , uttar pradesh , india . all the subjects ( females = 100 , males = 100 ) belonged to the age group of 1726 years . non - glossed lip stick color , white bond paper , cellophane tape , scissors , and magnifying lens were used for identification . length and thickness of the upper and lower lips were measured with sliding calipers . for all the lip prints , the thickness of the lip was measured at the center of the lip.results:overall accuracy by the examiner was found to be 81% ( out of 200 participants , 162 were diagnosed correctly ) and distribution of lip patterns showed a statistically significant difference between the genders ( p 0.05 ) . for the upper lip , the average difference in lip thickness found between males and females was 3.8 mm . however , the average difference for the lower lip between males and females was 2.9 mm.conclusion:in the present study , the lip prints of the subjects did not match with each other . the study reveals that lip prints behold the potential of determination of the sex of the person .

INTRODUCTION Classification of lip prints (Suzuki and Tsuchihashi, 1970) MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC3666211

current endeavours to eliminate lymphatic filariasis have been hailed as a best buy in global health ( ottesen , 2008 , e317 ) and the most effective pro - poor public health programme ( molyneux , 2003 , p. 1 ) . they are part of an international agenda to rescue the bottom billion through the control of neglected tropical diseases ( hotez et al . , 2009 , while there may be a degree of fundraising rhetoric in such statements , there is no doubt that lymphatic filariasis presents a major public health problem for large numbers of impoverished people . recent estimates suggest that around 120 million people living in tropical and sub - tropical environments are affected , with approximately 40 million displaying clinical signs of infection such as hydrocele ( swollen scrotum ) , lymphoedema / elephantiasis and a further 80 million people experiencing sub - clinical signs of infection ( taylor et al . , 2010 ) . strategies to eliminate lymphatic filariasis currently rely heavily on the mass distribution of drugs , free of charge , to all adults and children aged five or more living in endemic areas . the drugs being distributed are albendazole , in combination with either diethylcarbamazine citrate ( dec ) or ivermectin ; and recent reports suggest that more than 570 million individuals , residing in 51 countries where lymphatic filariasis is endemic , have received these drugs ( taylor et al . , 2010 ) . first , it is important to break the cycle of transmission between mosquitoes and human beings . anopheles , culex , aedes and mansonia mosquitoes are all capable of transmitting parasitic worms ( wuchereria bancrofti in africa ) that cause the disease , lymphatic filariasis , in humans . the mass distribution of drugs provides an opportunity to interrupt transmission , because the tablets have the effect of reducing the circulation of microfilariae ( the larvae of the parasitic worms circulating in the blood of infected individuals ) . second , elimination of the disease is thought to be achievable with the drugs mentioned above if a large enough proportion of the population swallow the tablets being distributed . the necessary rates of drug take - up have been variously estimated at 6580% over four to six years ( mohammed et al . , 2006 ) and 7090% over six or more years ( michael et al . , 2004 ) . in areas where the prevalence of microfilariae is greater than 10% , 2004 ) have suggested that coverage levels of 90% or more may be necessary . whatever the requisite coverage levels , multiple rounds of treatment are essential because the drugs currently being used do not kill the adult worms . instead , they reduce the circulation of microfilariae until the worms naturally die off , and it can take six years for this to occur ( michael et al . , 2004 ; bockarie & deb , 2010 ) . additional reasons for promoting mda include the fact that two pharmaceutical companies , glakosmithkline and merck , have agreed to donate the relevant drugs until lymphatic filariasis has been eliminated from the globe ( who , 2012 ) . the drugs , it is argued , can usually be distributed through existing national health mechanisms to volunteers in the targeted communities . they are also considered to be safe enough to be used without testing for infection , making it ethical , as well as cheap , to treat everyone . in sum easy - to - do and inexpensive health intervention ( molyneux , 2003 , p. 13 ) , a view that underpins the world health organization 's global programme to eliminate lymphatic filariasis . by 2008 elimination of the disease was anticipated to be a real possibility by 2020 ( end in sight for elephantiasis , bbc , 8th october 2008 , http://news.bbc.co.uk/1/hi/health/7659222.stm ) . continuing optimism is reflected in statements such as those by the who department of control of neglected tropical diseases in 2011 , suggesting the campaign is halfway towards eliminating lymphatic filariasis ( http://www.who.int/neglected_diseases/integrated_media/integrated_media_urogenital_lf_2011/en/index.html ) , and by those associated with the january 2012 joint london declaration on neglected tropical diseases ( ntds ) , in which partners pledged new levels of collaboration and announced a co - ordinated push to accelerate progress toward the eradication of lymphatic filariasis and the control or elimination of a range of other ntds ( http://www.dfid.gov.uk/documents/publications1/ntd%20event%20-%20table%20of%20commitments.pdf ) . the tanzanian national lymphatic filariasis elimination programme ( tnlfep ) is part of these global operations to eliminate the disease . in 2009 , it was reported that it had covered 34 districts in six regions , with 9.2 million people having been treated over a period of eight years ( malecela et al . , 2009a ) . efforts to eliminate lymphatic filariasis from tanzania are also part of the national programme to control neglected tropical diseases ( united republic of tanzania , 2009 ) . this programme was established in 2009 with a view to co - ordinating efforts to control or eradicate a variety of tropical diseases including schistosomiasis , soil - transmitted helminths , onchocerciasis , trachoma and lymphatic filariasis . it is recognized in reports and official statements that an integrated and holistic approach to lymphatic filariasis ( and other neglected tropical diseases ) is the ideal , and that mda should be combined with other methods . however , it would be fair to say that , in practice , the focus has primarily been on the distribution of donated drugs . in addition to the mass distribution of ivermectin and albendazole , it is intended that there should be vector control ( involving the use of bed nets and the reduction of breeding sites for mosquitoes ) ; lymphoedema management ; and hydrocelectomies ( involving surgery for scrotal swelling caused by the parasitic worm , wuchereria bancrofti ) . although the emphasis given to these latter components may be different in other locations , such as zanzibar ( mohammed et al . , 2006 ) and mafia ( malecela et al . , 2009a ) , at locations studied in this paper , vector control was receiving almost no attention , and lymphoedema management was very limited . a variety of epidemiological and health policy research has been undertaken on lymphatic filariasis in coastal tanzania ( see , for example , kolstrup et al . , 1981 ; meyrowitsch et al . , 1995 , 2004 ; nielsen et al . , 2006 ; rwegoshora et al . , 2007 ; simonsen et al . , 2005 , 2010 ; malecela et al . much of this research documents the prevalence of microfilariae as well as visible signs of infection such as swollen scrotums , legs and feet . ( 2010 ) , for example , reported that in kirare , a sentinel surveillance site located about 20 km from the regional capital of tanga , the overall prevalence of microfilariae among individuals over the age of one was 24.5% , and it was 53.3% for wuchereria bancrofti - specific circulating antigen . they also noted that 4.1% adults aged 20 years or more had elephantiasis of the leg and 32.8% adult men had hydrocele . these data were collected in september 2004 , immediately before the tnlfep began the first round of treatment . ( 2004 ) are correct , a daunting annual mda take - up rate of at least 90% would be required to interrupt disease transmission . go on to document the effect of three rounds of mass treatment with ivermectin and albendazole to adults and children aged five or more living in kirare . the reported findings suggest the uptake of drugs was less than the target rate , and that uptake declined over time . self - reported uptake fell from 82% in october 2004 to 79% in february 2006 and 69.9% in may 2007 . beneficial biological effects for the population were nevertheless observed . human infection with microfilariae declined , and however , the effects thereafter levelled off and transmission still occurred after the third round of mda , albeit at a lower level . concerns were raised about the length of time between rounds of treatment ( as they were longer than the recommended period of one year ) as well as the fact that mass treatment had limited influence on adult worm burden . the article concludes by highlighting the waning effect of mda and the need for careful monitoring and evaluation . this includes the need to make more accurate assessments of the number of people receiving and actually swallowing tablets . with respect to the latter point , the authors query the high rates of drug uptake recorded in official reports . this , in turn , raises the additional issue of whether or not the uptake of drugs in kirare represents the norm , or whether proximity to tanga town ( the location of the regional referral hospital and medical headquarters ) combined with the regular visits of tanga - based researchers , facilitated higher than average treatment coverage for the region . it is likely that this is the case as it would have been extremely difficult to have collected blood samples without a significant proportion of participants understanding the purpose of the research and the rationale for mass treatment . anthropologists are well - placed to assist with endeavours to assess the social and biological consequences of mass treatment for the control of neglected tropical diseases ( ntds ) such as lymphatic filariasis . to the authors ' knowledge , they have not , as yet , explored local responses to mass treatment programmes seeking to eliminate lymphatic filariasis . building on research undertaken in north - western , northern and south - eastern uganda on two other neglected tropical diseases , schistosomiasis and soil - transmitted helminths ( parker et al . , 2008 , 2012 ; parker & allen , 2011 ) , it addresses the following questions : is current enthusiasm for the elimination of lymphatic filariasis on the global stage shared by those on the receiving end of these programmes ? is it appropriate to assume that adults will be willing , if not grateful , to swallow tablets if they are provided free of charge , in the interests of maintaining or preserving their own health ? how does the social context in which mass distribution occurs influence the uptake of drugs ? the article is based on research undertaken in northern coastal tanzania from july to september 2007 and july to august 2011 . in 2007 , ten weeks of fieldwork were undertaken in the adjoining villages of mwembeni and jaira in pangani district , the small town of kigombe in muheza district and several other villages close to the town in this district . follow - up research occurred four years later over a period of five weeks . they were assisted by a group of four locally recruited researchers in 2007 and five locally recruited researchers in 2011 , all of whom were either secondary - school leavers or graduates who had been unable to find employment . research in mwembeni and jaira villages in 2007 involved participant observation in a wide range of settings , including visits to homesteads , attending local healing ceremonies , talking to villagers cultivating maize and cassava , and visiting local dispensaries and the district hospital . in addition , 30 open - ended , unstructured interviews were undertaken with local healers , health care providers and village elders ; and village meetings were held at various points during fieldwork to discuss the rationale for the research and to feed back provisional findings . to complement this work , a 20% random sample of households was generated from the 2004 ministry of health registers documenting the uptake of drugs for lymphatic filariasis . semi - structured interviews were then undertaken with one adult from each selected household . seventy - three semi - structured interviews were undertaken with adults in mwembeni village and 35 semi - structured interviews were undertaken with adults in jaira village . in july august 2011 , a further 118 semi - structured interviews were undertaken with randomly selected adults in mwembeni . in contrast to 2007 , it was not possible to use the ministry of health registers as a way of systematically identifying households in mwembeni as the records were no longer based on census data detailing the names of all residents living in the villages . instead , the registers were simply a list of names of people who had received treatment . a sample of households was thus derived by visiting every other household in each of the sub - divisions of the village . in addition , participant observation and fifteen open - ended , unstructured interviews were undertaken as well as an open meeting in the village to discuss key issues emerging from the research . in muheza district , for administrative purposes the area is divided into kigombe east ( and its sub - villages ) and kigombe west ( and its sub - villages ) . the peri - urban area of kigombe east runs along the edge of the sea . in addition , there are numerous villages , spread out over many kilometres , located to the north - east along the coast road running towards tanga town . the peri - urban area of kigombe west spreads inland from the coast road and includes more distant rural villages such as mchangani and ndonde . with the exception of the peri - urban area of kigombe west ( for which there was a household list produced by an american anthropologist , suzi krehbiel , who had lived in the area in 2006 ) , local registers were unavailable or incomplete . it was thus necessary to derive a sample of households by visiting every second home with a view to interviewing a similar proportion of people to that achieved in mwembeni and jaira . a total of 146 semi - structured interviews were undertaken in kigombe west ( including the sub - villages of mchangani and ndonde ) and kigombe east ( including the sub - villages of sinawe , gawani and the village of kitongoji ) in 2007 and 144 semi - structured interviews in 2011 . the methods employed were the same as those described for mwembeni and jaira in pangani district , including a similar number of unstructured interviews . research was also undertaken in 2007 in two villages located close to the district capital , muheza town : magila and bwembwera . here , 30 households were randomly selected in magila and 51 households were randomly selected in bwembwera . according to the lists of households kept by the village chairmen , this represented more than 20% of the estimated number of households . semi - structured interviews were then undertaken with one adult from each selected household . all semi - structured interviews undertaken in pangani and muheza districts elicited information about whether or not the adult being interviewed accepted free drugs for the treatment and prevention of lymphatic filariasis . information and ideas emerging from participant observation fieldwork and open - ended , unstructured interviews were subsequently used to interpret these quantitative data . swahili is the dominant local language in this part of tanzania , as well as being the national language . migrants into the region all spoke swahili , even when they also spoke their own languages ( such as makonde ) . the vast majority of semi - structured and unstructured interviews , as well as village meetings , were thus undertaken in swahili . finally , an endeavour was made to elicit the perspectives of village - based drug distributors and district officials involved in the mass drug administration programme for the control of lymphatic filariasis . official records monitoring the uptake of drugs by staff from the ministry of health were looked at wherever possible , and these were discussed with the relevant staff in english . mwembeni village is about 8 km from pangani town and lies on the main road that links muheza town to the coast . it is one of three villages that form part of madanga ward , and one of eight villages that form part of madanga division . according to census data published in 2006 , 1126 people live in mwembeni . the village is divided into four sub - divisions : mzia , mwembeni , makuyuni and nunda . it is socially diverse , with christians ( protestant and catholic ) and muslims ( sunni and shia ) living side - by - side . the christians are predominantly makonde , and the muslims are predominantly zigua and zaramo . there is inter - marriage between these groups , although tensions exist between muslim groups ( focused in particular on rival imams ) as well as between muslim and christian groups . it is not unusual for muslims to talk about the makonde as migrants or refugees from mozambique , in spite of the fact that they have lived in the village for two and even three generations . many of their homes are located at some distance from the village centres , in some cases in remote places . mwembeni village has one primary school and one small madrasa ( islamic school ) . it also has several mosques and churches ( both of the most basic variety ) . a small number of people have access to piped water from a tap every other day , and there are ten households with access to electricity , two of whom have a satellite dish . broadly similar characteristics apply to jaira village , which lies a few kilometres north of mwembeni . according to ministry of health data collected in 2004 , the majority of adults in jaira and mwembeni own or rent plots of land that they cultivate . others are employed on a temporary basis as farm labourers . a diverse array of crops coconut , orange , mango and banana trees are grown too , though most people do not have access to all these types of fruit trees . a small number of people fish for prawns in the river , and these are mainly sold in pangani town . it is important to note that very few people have regular , salaried work . historically , the large - scale production of sisal at nearby estates was an important source of regular employment , and explains the presence of so many makonde . however , decline in the global demand for sisal has led to a decline in production and the laying off of staff . as part of the surveys on drug uptake discussed below , adults living in mwembeni and jaira were asked what they considered to be the most serious problems facing them : 58% mentioned unemployment and kigombe is a small town that lies on the coastal road linking the town of tanga to the town of pangani . although it is geographically much closer to pangani , it is actually part of muheza district . access to the sea provides fishing opportunities to those with access to nets and boats , but the majority of people rely upon the cultivation of cassava , maize and rice to survive , just as they do in mwembeni . fifty - three per cent of surveyed adults highlighted unemployment as the most serious problem they faced compared with 31% who mentioned inadequate health facilities . there are several schools in kigombe , including a madrasa , and there are also several mosques and a government dispensary . the population is mostly muslim , but in common with mwembeni , there is a christian minority , generally living a little further inland at the edges of the sisal fields . also like mwembeni , there are divisions among the muslims , but of a more intense nature . radical islam has a presence ( with some openly sympathetic to al qaida in 2007 ) . there are also divisions between more orthodox sunni muslims and muslims who are members of the sudanese ansar sect . in contrast to mwembeni , kigombe is characterized by the amplified call to prayer from rival mosques at night time . other divisions relate to political activism , with the local chairman being a member of civic united front ( cuf ) , a political party opposing the government . before addressing the specific issue of drug uptake for the treatment and prevention of lymphatic filariasis , it is helpful to note that the study populations were found to draw upon a broad range of ideas to understand illness and misfortune . interpersonal and spiritual aspects of well - being are often used to interrogate biomedical understandings , with many kinds of illness being discussed in relation to the swahili word uchawi . witchcraft , but it does not necessarily have the same connotations as the english word . the majority of people interviewed in depth talked about uchawi as being a constant threat , and many were surprisingly open about the specific individuals who they suspected were most likely to use it . even in the semi - structured interviews , which were inevitably less probing , large numbers of people stated that uchawi presented a problem . notions about interpersonal and spiritual causes of affliction are also connected with beliefs about and experiences of spirit possession . those local healers with high status were all found to be members of swahili possession cults . in common with giles ( 1987 , 1999 ) , it became apparent that , unlike most possession cults in other parts of africa , these cults are closely connected with local hierarchies , and are expensive to join . possession is viewed as something dangerous and frightening , but paradoxically , also carries high status and the possession cults operate as a parallel means of interpreting the spirit world to that of islam . indeed some of the practices observed were strikingly un - islamic ( such as the ritual drinking of sheep blood ) . to the authors ' knowledge it is also important to note that considerable emphasis was placed on the training of healers at public sances . for example , more than a dozen healers were present at a ritual that went on for several days at one of the villages of kigombe and half a day was allocated to discussions between healers attending the ritual and those who had become new members of the cult and would in the future be acting themselves as healers . emphasis was placed on the appropriate relationships they should have with their patients , implicitly contrasting them with the kinds of treatment expected at clinics . their treatments involved a combination of local remedies , concocted from locally available substances , with spiritual and moral interpretations of affliction . the findings of respected healers carried weight , and were not necessarily complementary with biomedical interpretations and treatments , including those for lymphatic filariasis . by july 2007 , three rounds of treatment had been distributed , free of charge , over a four - year period for lymphatic filariasis . these took place in 2004 , 2006 and 2007 . in both districts , registers were distributed by staff from the ministry of health to village - based drug distributors prior to the first round of treatment in 2004 . this enabled a detailed record to be made of the names and ages of people living in each household with a view to keeping a year - on - year record of the proportion of adults and children receiving drugs for the treatment and prevention of lymphatic filariasis . in mwembeni village , distributors were found to have kept meticulous records of the number of adults and children aged five and above who took the drugs for lymphatic filariasis . according to these registers , 59% of adults and children eligible for treatment received the drugs in 2004 ; whereas 46% of them received treatment in 2006 and 34% received treatment in 2007 . in short , data derived by selecting a 20% random sample of households and then interviewing one adult from each selected household also indicated a decline . that is , 71% of adults interviewed in mwembeni said that they received treatment in 2004 ; whereas 49% received treatment in 2006 and 35% received treatment in 2007 . table 1.uptake of drugs for the treatment and prevention of lymphatic filariasis among adults in pangani district , tanga region , 20042007200420062007drug uptake among adults and children in village registersmwembeni village 202 ( 59%)402 ( 46%)306 ( 34%)jaira village 263 ( 55%)164 ( 43%)160 ( 42%)self - reported drug uptake among interviewed adultsmwembeni village , n=7553 ( 71%)37 ( 49%)26 ( 35%)jaira village , n=3419 ( 56%)17 ( 50%)13 ( 38%)ain mwembeni village , the total number of people listed in the drug distributors registers that were eligible for treatment was 345 in 2004 , 869 in 2006 and 901 in 2007 . the fact that fewer people appeared to be eligible for treatment in 2004 can almost certainly be attributed to the fact that some of the registers had been lost.bin jaira village , the total number of people listed in the drug distributors registers that were eligible for treatment was 475 in 2004 , 386 in 2006 and 386 in 2007 . the fact that fewer people appeared to be eligible for treatment in 2006 and 2007 , compared with 2004 , is probably attributable to the fact that some of the registers had been lost . uptake of drugs for the treatment and prevention of lymphatic filariasis among adults in pangani district , tanga region , 20042007 in mwembeni village , the total number of people listed in the drug distributors registers that were eligible for treatment was 345 in 2004 , 869 in 2006 and 901 in 2007 . the fact that fewer people appeared to be eligible for treatment in 2004 can almost certainly be attributed to the fact that some of the registers had been lost . in jaira village , the total number of people listed in the drug distributors registers that were eligible for treatment was 475 in 2004 , 386 in 2006 and 386 in 2007 . the fact that fewer people appeared to be eligible for treatment in 2006 and 2007 , compared with 2004 , is probably attributable to the fact that some of the registers had been lost . the table also shows that very similar findings were recorded in neighbouring jaira village . the jaira registers suggested that 55% received treatment in 2004 ; 43% received treatment in 2006 and 42% received treatment in 2007 . in common with mwembeni , a 20% random sample of households was taken from the 2004 ministry of health drug distribution registers . one adult was interviewed from each selected household , and data emerging from these interviews also conveyed a decline in the uptake of drugs . that is , 56% adults received treatment in 2004 , 50% received treatment in 2006 and 38% received treatment in 2007 . ministry of health registers documenting the uptake of drugs in kigombe and surrounding villages could not be located . however , data collected in the course of undertaking semi - structured interviews suggested a downward trend . table 2 shows that 47% of those interviewed in kigombe said they received treatment in 2004 , 45% received treatment in 2006 and 22% received treatment in 2007 . a different picture emerged in the northern part of muheza district . in the village of bwembwera , interviews indicated that 47% received treatment in 2004 , 69% received treatment in 2006 and 65% received treatment in 2007 ; whereas in magila , 40% received treatment in 2004 , 43% received treatment in 2006 and 70% received treatment in 2007 . table 2.self-reported drug uptake for the treatment and prevention of lymphatic filariasis among adults in muheza district , tanga region , 20042007village200420062007kigombe , n=14668 ( 47%)66 ( 45%)32 ( 22%)bwembwera , n=5124 ( 47%)35 ( 69%)33 ( 65%)magila , n=3012 ( 40%)13 ( 43%)21 ( 70%)akigombe includes kigombe east and kigombe west . kigombe east includes the sub - villages of sinawe , gawani and jitingeni and kigombe west includes the sub - villages of ndone and mchangani . self - reported drug uptake for the treatment and prevention of lymphatic filariasis among adults in muheza district , tanga region , 20042007 kigombe includes kigombe east and kigombe west . kigombe east includes the sub - villages of sinawe , gawani and jitingeni and kigombe west includes the sub - villages of ndone and mchangani . in july august 2011 , 118 semi - structured interviews were undertaken with a random sample of adults in mwembeni , pangani district , to assess the proportion of adults that had received ivermectin and albendazole for the treatment and prevention of lymphatic filariasis since 2007 . as indicated in table 3 , the self - reported uptake of drugs was below 50% for the period 2008 to 2010 . taking a sample of randomly selected households near the trading centre , seventeen adults were interviewed , with 70% of them stating they had received the tablets . follow - up research was also undertaken in kigombe town and surrounding villages during this time . one hundred and forty - four semi - structured interviews were undertaken and table 3 shows that the self - reported uptake of drugs remained below 50% from 2008 to 2010 . table 3.self-reported drug uptake for the treatment and prevention of lymphatic filariasis among adults in mwembeni , pangani district , and kigombe , muheza district , 20082010village200820092010mwembeni , n=11826 ( 22%)45 ( 38%)56 ( 48%)kigombe , n=14445 ( 31%)57 ( 40%)49 ( 34% ) self - reported drug uptake for the treatment and prevention of lymphatic filariasis among adults in mwembeni , pangani district , and kigombe , muheza district , 20082010 official statistics submitted to the ministry of health from pangani district differ from the information recorded in the ministry of health village drug distribution registers . that is , the overall uptake of drugs for lymphatic filariasis among adults and children more than five years old was reported to be 67.6% in 2004 , 78% in 2006 , 76.5% in 2007 and 75.2% in 2010 . aware of the possibility that these aggregate figures may reflect much higher levels of drug uptake in other parts of the district , a decision was made in 2007 to go through all the available official village - based , drug distribution registers for pangani district in order to double - check the accuracy of the official figures . in addition to the registers for mwembeni and jaira , registers were found for six of the 33 villages that constitute pangani district . these villages were : barabani , chakaloboko , mahakamani , boza , kimanga and msaraza bushiri . not all registers for these villages were found to be complete for all three recorded rounds of drug distribution , but according to the registers available , drug uptake fell short of the sustained high coverage levels that are required to eliminate lymphatic filariasis . to be more specific , the uptake of drugs among the 4755 adults and children listed in the available registers as eligible for treatment in 2004 ranged from 49% in one village to 93% in another village , and the average uptake for all eight villages was 65% . however , the uptake of drugs declined in all villages except one in 2006 . that is , uptake ranged from 81% to 35% , and the average uptake among the 7823 adults and children listed as being eligible for treatment was 46% . in 2007 , there were further declines in the majority of villages . uptake ranged from 81% in one village to 7% in another village and the average uptake was 34% among the 6436 adults and children listed as being eligible for treatment . the average figure for 2004 was thus very similar to that in the officially submitted report , but the high drug uptake reported in 2006 and 2007 in the official records submitted to the ministry of health did not correspond with the drug uptake data recorded in the village registers . while it is possible that the uptake of drugs is higher in other parts of pangani district , this is an unlikely scenario . many of the villages for whom drug distribution registers could not be found were located south of pangani town . this part of the district is often inaccessible to district staff as it involves crossing the pangani river by a ferry that is prone to mechanical breakdowns . technical difficulties aside , budgetary constraints at district headquarters restrict the frequency with which staff can visit villages and facilitate the distribution of drugs not least because many of the villages are located a considerable distance south of the river . as a result , it was much harder to elicit comparable data about the uptake of drugs in muheza district . in 2007 and 2011 , considerable efforts were made to talk to district officials in muheza as well as associated officials at a ward , village and sub - village level , but no ministry of health registers could be located in 2007 , and only a small number could be located in 2011 . in addition , it was difficult to make much sense of the information presented in the registers that could be located as they only provided a list of the names of people that had received treatment in 2010 . in other words , in common with the registers located in mwembeni in 2011 , it was not possible to use the information to assess the proportion of people in any one village that had received treatment . nevertheless , summary statistics compiled at a district level ( and subsequently presented as part of a regional report submitted to the ministry of health in dar es salaam in 2009 ) , recorded an uptake of drugs in the district of 81% in 2006 , 76% in 2007 and 81% in 2008 . the neglected tropical diseases co - ordinator for muheza district also reported coverage of 83% in 2009 and 83% in 2010 whilst acknowledging that the uptake of drugs was less in kigombe as official returns suggested an uptake of 69% in 2009 and 65% in 2010 ( personal communication , august 2011 ) . while these figures are lower than the 100% uptake reported to the research team by a district official in muheza in july 2007 , they are still much higher than those recorded in the course of interviewing randomly selected adults at selected villages in muheza district . discrepancies between the figures derived from semi - structured interviews , the official district figures , and the figures in the village - based registers raise important questions about the relative merits of the methods employed to elicit information on drug uptake . with respect to data arising from semi - structured interviews , is it really possible for large numbers of adults to accurately remember whether or not they have received a particular combination of drugs over an extended period of time ? is it possible that some informants muddle up the years that they received them and the years that they did not ? is it appropriate to assume that informants receiving drugs during mda actually swallowed them ? whatever is done to alleviate anxieties and ensure that those being interviewed recount events as accurately as possible , is it possible for informants to try and second - guess the correct answer to be polite or to just make the researchers leave them alone ? identical questions emerged whilst undertaking research on social responses to mda for the treatment of schistosomiasis and soil - transmitted helminths in northern and south - eastern uganda ( parker et al . , 2008 , 2012 ; parker & allen , 2011 ) . here it was found that those interviewed clearly recalled treatment , partly because praziquantel tablets , which are used to treat schistosomiasis , are unusually large , pungent and unpalatable . in addition , the free distribution of biomedical drugs to treat adults is a rarity and not something easily forgotten . indeed , it is widely commented on and the issue is less whether or not people remember , and more whether or not they are willing to say what happened to the drugs once they had received them . in some cases it became clear that instead of swallowing them immediately , they had kept all or some of their dose for future use if they felt ill , or to give to relatives and friends . the situation was found to be slightly different with respect to mda for lymphatic filariasis in coastal tanzania , not least because the drug , ivermectin , is small , easy to swallow and does not have an unpleasant smell . nevertheless , few people struggled to recall whether or not they had received free treatment partly because it was so unusual to offer free medicines to adults and partly because it was considered to be very strange indeed to ask all adults to swallow medication , irrespective of whether or not they felt ill . in fact , the vast majority of people interviewed were remarkably forthcoming about their reasons for accepting or rejecting treatment and , more often than not , they asked pertinent questions about the rationale for the control programme that shed further light on why , for example , some people accepted treatment in the early years of the programme and subsequently declined treatment ; while others declined treatment but subsequently accepted treatment for one or two years , before deciding to reject treatment again . in general , data recording the uptake of drugs from semi - structured interviews undertaken at randomly selected households are , in most cases , likely to more accurately convey the proportion of adults that received the tablets than the data embedded in the official ministry of health registers by village - based drug distributors . first , villages can be quite dispersed and drug distributors were sometimes found to be unaware of all people living in the village . second , social divisions within a village can often affect the kind of people recorded . in mwembeni , for example , the long - standing differences between muslims and christians were found to have influenced the number of people registered . refugees , or even not real people. third , short - cuts are often taken in the time - consuming job of handing out tablets . close discussion with the distributors and people receiving drugs revealed that the protocol of observing consumption was often not followed . in several instances , it was found to have been largely set aside , in a similar manner to the practice noted above with respect to praziquantel distribution in uganda . as a result , it is not known if those who are recorded as having taken the drugs have actually swallowed them . thus , drug uptake data in the village - based distribution registers may overestimate , rather than underestimate , actual consumption . this is not to suggest that the data in the ministry of health registers are always inaccurate . on the contrary , some drug distributors went to great lengths to carefully record the names , ages and sex of the people they gave drugs to . however , the consistency of registration between distributors was found to vary ; and it was often impossible to judge the quality of the recorded data without checking them . in most cases , they provided , at best , a partial picture of the proportion of people in a specific geographical area that had received treatment . a more worrying issue is that the summary statistics compiled at a district level on drug uptake suggest much higher levels of drug uptake than data eliciting drug uptake among adults in the course of interviews . while the latter data only refer to adults , it is likely that they provide a useful proxy of the extent to which mda programmes reach adults and children at the selected study sites . the reasons are straightforward : mass treatment occurs through village drug distributors , rather than school teachers ; and if adults are willing to receive ( and possibly consume ) tablets for themselves , then they are almost always willing to receive tablets for their children . similarly , if they reject medication for themselves , then they are likely to reject treatment for their children . it is also worth noting that in those instances in 2004 and 2005 when village drug distributors recorded the numbers of people living in a village on a household - by - household basis in the ministry of health registers and whether or not each person had received drugs , there was a similar discrepancy with drug uptake being much lower in the registers than the aggregate figures recorded at a district level . senior district officials , when asked to comment on the discrepancies , were quick to set aside the higher figures . one of them disarmingly observed : people are just sitting down and cooking the numbers. he went on to express frustration with the treatment programme by saying this programme has no legs. this was a literal reference to the shortage of fuel and other resources enabling staff to spend the requisite time in villages explaining the rationale for mass treatment . he was , in short , not surprised by the lower drug uptake figures emerging from field - based research . this view was shared by others working at district headquarters . in short , there is no doubt that the uptake of drugs is low at the selected study sites ( and rates of take - up in other highly endemic coastal villages in pangani and muheze districts are likely to be similar ) . the low uptake of drugs can not be attributed to a lack of awareness of the signs and symptoms of lymphatic filariasis . while there is no swahili word for the disease , two of the major clinically recognized signs of infection , swollen scrotums and swollen limbs , are perceived locally as separate afflictions , with the former being referred to as mabusha and the latter as matende . both are feared , even if they are not considered as life - threatening as malaria or hiv / aids . adults participating in the surveys on drug uptake were asked what they thought were the major illnesses affecting local people . it was mentioned by 64% of those interviewed at study sites in pangani and 78% of those interviewed at study sites in muheza , whereas hiv / aids was mentioned by 29% of adults in pangani district and 19% of adults in muheza district . mabusha was mentioned by 27% and 24% in pangani and muheza districts respectively and matende by 12% and 15% although muscle or leg pain was also mentioned by 26% and 20% of adults respectively in these two districts . in more open - ended discussions , mabusha , in particular , was spoken about as a constant threat for men . for example , a 27-year - old man from jaira village explained that mabusha is a torturing disease : [ it ] makes you sad , unhappy , everyday ( all such quotes in the article have been translated from swahili ) . another man , aged 31 , and also from jaira said , i feel so ashamed , especially with those i respect . i have access to women but they always say : i do n't want you , because you will give me the infection. others , including a 52-year - old man from kigombe , complained that people talk about you when you are not there . it makes you fearful and anxious no woman will want you if you have mabusha unless they are a witch and they want your money . in some cases , the distress , misery and desperation was so intense that men tried to operate on themselves . in 2007 , for example , a 40-year - old man from mwembeni told us that he had operated on himself twice to try and drain the fluid from his scrotum . tragically , he died in 2008 , reportedly whilst trying to perform the operation on himself for a third time . attitudes to matende tended to be more stoical , but the worst cases observed were shocking , with afflicted individuals , mostly women , being hidden away out of site . given that mabusha and matende are widely recognized to cause sickness and suffering , one might assume that the provision of free treatment would be welcomed . the rest of this section demonstrates that there is no single explanation for the low uptake of drugs . rather , a combination of social , economic and political reasons explain the low uptake of drugs at the different study sites . these include : fear of treatment ( attributable , in part , to a lack of trust in international aid and a questioning of the motives behind the distribution ) ; divergence between biomedical and local understandings of lymphatic filariasis ; insufficient and inadequate communication about the rationale for mass treatment ; and too great a reliance upon too few volunteers to distribute drugs within villages . mass drug administration for mabusha and matende generated anxiety and scepticism among the majority of adults , with informants questioning the motives behind the free distribution of drugs by the tanzanian government and international agencies . a young man from jaira , for example , said : there is something strange going on . if the government want to help us , they should distribute malaria drugs for free , not matende and mabusha. similarly , a 40-year - old man from mwembeni commented that , there is no sign [ on the drug ] that it is for mabusha or matende you have to trust in the government to swallow the tablets trust matters a lot . if you do n't trust them , you ca n't swallow the drug the programme does n't come with enough knowledge. meanwhile , in kigombe , a man in his 50s justified not taking the tablets in the following way : in this village we cry for water they haven't brought water for free , but they 've brought drugs for free . why ? these free drugs have to be for an experiment that is why i reject them. these kinds of comments were typical in locations with low drug uptake and highlight the fact that the rationale for distributing tablets for mabusha and matende , free of charge , was poorly understood . acutely aware that clinic - based biomedical practitioners tend to diagnose infections before administering drugs , and confused why such an approach was deemed unnecessary by the tanzanian government in this case , rumours circulated about the a very prevalent concern was the idea that the drugs had been designed to reduce population growth . as one man in kigombe put it : they are lying about this drug . it is not for mabusha or matende [ it is ] a family controlling pill . younger men discussing this issue tended to emphasize the idea that the tablets reduced their sexual energy . for example , at a village meeting in mwembeni , a young man summarized the views of others when he said , they are afraid they say if you are infected [ and you ] take the drug then your penis will never erect again. it was a view echoed at a comparable group discussion at kigombe , where a man in his mid-30s proclaimed that [ people from the ] rich countries send us this drug to reduce our malehood energy and to make us infertile , because the wazungu ( white people ) were fed up giving us help every day. for others , the focus was more on the way the tablets might be intended to prevent conception , a view that was probably supported by the reluctance of some drug distributors to give the tablets to women who said they were pregnant . for example , a man in his 60s living in mwembeni commenting on local views said , they say if you take the drug , you will never give birth they believe the government has failed to convince them to enter into family planning. women were particularly likely to say something comparable . we know women in europe have two children and their intention is to make africans like europeans . they think we ca n't manage so many children , that we ca n't provide proper education , health care and home environment. concerns about infertility were also connected with the experiences of some of those who had swallowed the drugs . a man in his 30s from mwembeni , for example , described how he took the tablets in 2006 and experienced a strong fever with rashes . he went on to say that he did not take the drug again as he feared the side - effects . others expressed concern that the drugs exacerbated existing symptoms , and even infected people with mabusha and matende . a woman from kigombe , for example , said that she did not take the drugs in 2004 as she was pregnant . seen people infected by those drugs in 2004 there were some who got body pain and fever . if i took those drugs , i would be infected too . in a similar vein , an elderly woman from mwembeni said : they were afraid that if they took the tablets the busha would get bigger, and a man from kigombe said : they are afraid that if they take the drug they will get matende or mabusha . there were , in addition , stories that were even more alarming . a young man from mwembeni , for example , said : they fear the drugs will kill them . he was not the only one to mention this . he went on to explain that , most people do not receive reliable information and knowledge about the drugs. these last remarks relate to prevalent doubts about drug efficacy . this is hardly surprising as few , if any , people were aware that it was possible to be infected and asymptomatic and almost all of those with mabusha and/or matende complained that the drugs were not making much or any difference . for example , a man in his 60s from kigombe had lived with mabusha since his mid-40s and took the treatment in 2004 , 2006 and 2007 , but declined free treatment in subsequent distributions . he explained that , the drugs make no difference the weight of my testicles disturbs me. his experience was shared by a younger man who had accepted treatment in 2004 , but subsequently refused it . some other people i know took the drug and then got mabusha , so they did n't take it again either. these perspectives were widely shared . a woman in her 30s with matende who had also taken the drugs only once summed up attitudes when she said : we are getting no relief from the medicine the drugs are not working. widespread fears , anxieties and rumours emerge , in part , from villagers making their own empirical observations about the side - effects associated with treatment and the limited visible efficacy of the drugs . they also draw attention to the disjuncture between biomedical understandings and responses to lymphatic filariasis and local understandings and responses to mabusha and matende , which are perceived locally as entirely separate afflictions . to quote a local healer ( mganga ) from pangani : some people have both mabusha and matende but they do n't come simultaneously . the fact that mabusha can be operated on and matende can not be operated on , proves that they are different diseases . the majority of people do not attribute a single cause to either mabusha or matende . indeed , it is not unusual for three or four causes to be mentioned , and for each to be accorded equal weight . this sub - section discusses local understandings and responses to mabusha and matende . in so doing , it becomes apparent that these locally recognized afflictions are understood in a context where virtually everyone , including health care workers at government dispensaries and health centres , draws upon a pluralistic approach to diagnosis and treatment to understand sickness and misfortune . it is also worth noting that discussions about mabusha , and to a lesser extent matende , can be infused with moral overtones , with spirit mediums sometimes threatening those who are thought to be behaving badly by saying that they will throw a busha. in madanga division , for example , there was a ceremony associated with the local possession cult called mwaka koga , part of which was held at a sacred well on the outskirts of a village . people gathered spoke to the spirits in the well . at one point in the ceremony , the spirit medium said : protect us , cure us and treat us so that we can be healthy and have peace and happiness in our lives . there are some who see those who come to us as barbaric , uncivilized and ignorant ; and they see themselves as educated , civilized and more religious they only believe in one god [ to them we say : ] give them bushas and matende and never cure them , until they come here and beg for forgiveness. the majority of adults are unaware of any relationship between mosquitoes and mabusha . of the 335 adults interviewed whilst undertaking drug uptake surveys in pangani and muheza districts in 2007 , just 11% attributed mabusha solely to the bites of mosquitoes . while a small proportion of informants attributed mabusha to the bites of mosquitoes and other causes ( notably acts of witchcraft , sex , hernia , dimensions of diet , acts of god ) , the vast majority of informants either did not mention mosquitoes as a possible cause or remained unconvinced about their role in transmission . on one occasion , during a village meeting , a female sub - village leader took issue with the biomedical explanation being put forward by a health worker about the aetiology of mabusha and said : we can fight to the death , but you will never get me to believe that it is not caused by sex with a menstruating woman! the reference to sex is important here . it highlights the fact that mabusha is widely talked about as a sexually transmitted disease . the blood is full of vidudu ( micro - organisms ) god created menstruation to clean the woman from vidudu but these vidudu get into a man if they have sex when she is bleeding [ and it leads to mabusha]. a muslim elder added , the vidudu in the blood passes into the penis and it can lead to mabusha. others agreed with this point , but emphasized that it was only possible to get mabusha from a menstruating woman if the sex occurred outside of marriage . thus , a woman from mwembeni said , if you steal someone 's wife , who is protected by the healer , then you get mabusha with vidonda ( severe rashes on the penis), and a man from kigombe said , if you have sex with a prostitute that is menstruating , you can get mabusha you see , they will never tell you that they are menstruating as they want the money. in addition to sexual intercourse , other explanations for mabusha include one or more of the following : a lack of food , eating too many coconuts and/or drinking immature coconut juice , being bitten by mosquitoes and acts of god . hernia , too , is closely associated with the occurrence of mabusha . a 62-year - old male , for example , commented : a hernia is a disease . inside the hernia oil or water from inside the intestines pass down into the testicles and make a busha. similarly , a local mganga said : it catches you at the waist the muscles around the waist can squeeze the hernia , and the fluid goes into the testicles, and a young man , speaking at a village meeting , said : if you have a hernia , your testicles swell but it does n't mean that it 's hydrocele [ although ] most people think that it 's not possible to have a hernia and not have busha . ideas about witchcraft also play a central role in local understandings of the aetiology of mabusha . here , it is helpful to note that different kinds of mabusha are recognized locally , with some kinds being more overtly linked to acts of witchcraft than other kinds . two of the most widely recognized kinds of mabusha are : busha la maji and busha la mafuta . busha la maji is thought to be effectively treated by surgery at hospital as the water can be drained away , whereas busha la mafuta is not thought to be treatable in a hospital setting . as one 70-year - old male said : even if the fatty oil is scraped out , it just fills up again . others spoke of busha la mkoba and busha la uchawi . with respect to busha la mkoba , it is interesting to note that the word mkoba is swahili for woman 's handbag and that witches are sometimes thought to carry the tools of their craft in their testicles . as one mganga said : mgangas who hide their tools in a mkoba are not mgangas . they are witches , but they exist. busha la uchawi translates as bewitching busha and a local mganga described it in this way : in the village , if you have a conflict with a man in the village , he will bewitch you there are some diseases that can not be cured by biomedicine , only by herbs . many local mgangas and other villagers talked about their ability to make mabusha . a 35-year - old woman from kigombe , for example , said : if you have a quarrel with a person , they can take revenge and punish you by making mabusha there is nothing you can do no drugs can help. it is important to note , however , that several mgangas did not concur with the view that mgangas were capable of throwing a busha. as one mganga from madanga pointed out : mgangas sometimes say they can throw a busha , but this is not the work of a mganga . the work of mgangas is to cure , not to make sickness. whatever the role of mgangas , it became apparent that the majority of informants linked the occurrence of some kinds of mabusha to acts of witchcraft , with the type of mabusha influencing the type of treatment sought . moreover , mgangas are usually consulted to determine the cause of mabusha , and if it is deemed to be busha la mafuta , then biomedical treatment is avoided . busha la maji is thought to be treated by doctors and most mgangas recommend biomedical treatment , as they do not think they can treat anything but the early signs of mabusha such as fever . it is important to note , however , that some informants did not differentiate between types of mabusha . for example , a 63-year - old christian makonde in mwembeni remarked that : it is a shameful disease and you ca n't live with it you must be operated on as quickly as possible no local mganga operates on mabusha. similarly , a muslim man from kigombe observed that , the best [ thing to do ] is to go to hospital for an operation it is difficult for a mganga to treat mabusha because he has no equipment and no knowledge of what to do. in general , it was found that there is considerable demand for surgical treatment . however , many men do not go down this route as it normally costs approximately 100,000 tanzanian shillings ( about 40.00 ) and few have the necessary income to pay for it . an exception is when the operations have occasionally been offered free . in 2007 , for example , names of individuals who stated that they had hydroceles and wanted an operation but could not pay for it were collected at the field sites . two hundred operations were subsequently carried out on some of these men as well as other individuals at a surgical camp in pangani district with special funding in 2008 ( malecela , 2009a , p. 55 ; personal communication by email on october 19th 2007 ) . partly as a consequence , scores of others needing operations made themselves known to the research team in 2011 ( roughly 1:10 men in mwembeni ) , although many stated that , even if the operations are free , they would find it hard to be treated , because they could not afford not to work for six months after the operation . to quote a 30-year - old man from mwembeni : [ they tell us ] you ca n't do masculine ( i.e. heavy ) jobs for six months , but how will our children eat then ? what we do to earn a life is tough work if you rest for six months , your children will die. others are afraid to be operated on . one man , reflecting on the fact that his friend showed no inclination to get treatment from the hospital , said : he has decided to tie a tight rope around his testicles to try and prevent them from swelling. others are afraid that they will die if they are operated on . this fear relates to anxiety as to whether they have busha la maji or busha la mafuta as the latter needs to be treated by a mganga and , more significantly , treatment at a hospital is not only inappropriate , but can lead to death . to quote a mganga from mwembeni : some mabusha is caused by uchawi if you go to a hospital for an operation and it is caused by uchawi , you will die immediately. in common with mabusha , villagers attributed matende to a number of different causes . it was not unusual for several explanations to exist side - by - side and for each one to be deemed of equal importance . matende , for example , was often said to be an inherited condition as well as being caused by mgonzo fever and/or the bite of a mosquito . in contrast to mabusha , the majority of adults at all study sites said that they did not know what caused matende , but mgonzo fever was widely recognized as an early sign of matende . it is very different to the fevers associated with malaria , and one male informant described it in the following way : when it catches you , you will be seriously ill , to the extent that you urinate unconsciously , shiver and speak nonsense . two or three days later , the swelling goes down and the skin peels off . he or she might be ok for a year [ but ] then they will experience the fever again , until it becomes tende it is best treated with a painkiller. similarly , a muslim woman from kigombe east said : homa mgonzo can lead to matende . [ it ] is very strong you feel very , very cold and shiver all over the fever goes into the body 's muscles and the effect of this is that it goes into the legs and feet and leads to swelling . some people can get swellings in their arms too. various causes are attributed to the occurrence of mgonzo , including open wounds and sexual intercourse . the logic is clear : i cut my foot while i was farming and , soon afterwards , i got mgonzo . it was not [ an ordinary ] homa ( fever ) as the head does not hurt if you have homa and my head was hurting . some people also get matende from mosquitoes , but i didn't, ( muslim woman from kigombe who has had matende for many years ) ; mgonzo is a strong fever that leads to a swelling of the legs . i think i gave it to her through sexual intercourse, ( christian makonde man from mwembeni ) . in contrast to mabusha , a sizeable number of informants mentioned mosquitoes as the sole cause of matende ( for example , 37% in kigombe west in 2007 ) . maybe the mosquito has poison inside it and this is why the legs swell, ( a man in his 50s from kigombe east ) ; there is a mosquito called culex when that mosquito bites someone with matende and then bites you , you will get matende, ( a man in his 80s from kigombe west ) . nevertheless , like any other affliction , such interpretations were a possible explanation , often in combination with others . here people were jealous of her and they made uchawi ( witchcraft ) to give her matende, ( 60-year - old man from mwembeni ) ; when you have a conflict with someone , that person will go to a local mganga and make matende by using spiritual force, ( 34-year - old woman from kigombe ) ; mchango it is in your stomach , it eats the food you take in . bad mchango causes minyoo , busha and matende , together with safura ( hookworm), ( 65-year - old man from kigombe ) ; i got matende by stepping over uchawi ( witchcraft), ( 56-year - old woman from mwembeni ) . i think it is caused by a mosquito , but many people think it is inherited. if your father has it , then it can be passed on to sons , daughters , grandchildren like having twins. if the parent has twins , then anyone in the family can have them. my wife has matende in her right leg and her mother suffers from it too . she inherited the condition. although it is less common with respect to matende to assert a witchcraft explanation , the view is sometimes expressed that it is possible to differentiate between the type of matende caused by a mosquito and the type of matende caused by witchcraft . the following quotes are a good examples of the range of perspectives frequently exchanged among groups of people sitting together with an afflicted person and commenting on the problem : there is no treatment for normal matende , but witch matende can be treated by mgangas ( local healers). women can stop matende by wearing several pairs of underwear to stop it spreading. inside the matende is oil if you go to the hospital , they can remove the oil and the swelling will come down. the normal matende can be cured , and the swelling goes down , after treatment at the hospital . the other kind does not go down with hospital treatment. it has been argued that social mobilization and advocacy is a key component of effective mda for lymphatic filariasis in tanzania ( malecela et al . , 2008 , this is said to comprise everything from radio spots to posters , film shows and the use of mobile megaphones . all this activity is reported to occur in swahili and to have been incorporated into a set of lymphatic filariasis control programme principles. while these activities may well have occurred at some locations , it is also the case that they either have not occurred or they have had a minimal effect at the field sites where this research occurred . as a result , very few people understood the rationale for distributing drugs , free of charge , to adults and children . staff employed at health centres , as well as village - based drug distributors , were thus genuinely confused as to whether the drugs were designed to prevent infection or treat existing infections . with few exceptions , they had no idea that it was possible to be infected and asymptomatic and they were at a loss about how to answer frequently posed questions . these included variations of the following , all of which were posed at village meetings and/or during interviews in 2007 and 2011 : why should i take the tablets when i feel well ? why do the tablets need to be taken by everyone once a year ? why should i take the tablets again as i took the tablets last year and i still feel ill ? why should i take the drugs as i do not have mabusha or matende ? i took the drugs last year as i have mabusha , but the swelling did not go down . how can these drugs prevent mabusha when it is caused by witches and spirits ? why is there an operation for mabusha , but not matende ? why do some people get fevers and others swollen bodies after swallowing the tablets ? why do drug distributors allocate the number of tablets according to height rather than weight ? why are europeans manufacturing tablets for mabusha and matende when they do not suffer from these diseases ? why should i take the tablets when i feel well ? why do the tablets need to be taken by everyone once a year ? why should i take the tablets again as i took the tablets last year and i still feel ill ? why should i take the drugs as i do not have mabusha or matende ? i took the drugs last year as i have mabusha , but the swelling did not go down . how can these drugs prevent mabusha when it is caused by witches and spirits ? why is there an operation for mabusha , but not matende ? why do some people get fevers and others swollen bodies after swallowing the tablets ? why do drug distributors allocate the number of tablets according to height rather than weight ? why are europeans manufacturing tablets for mabusha and matende when they do not suffer from these diseases ? at all sites there was an eagerness to engage in debate and discussion about these issues , and innumerable complaints that the drugs were being given out by local distributors who could not explain how they worked or exactly what they were for . as one young woman from kigombe put it : there is no information about why you should take the drugs , what the drugs are for and other necessary information to convince people to take the drugs. with so many unanswered questions , it is hardly surprising that people questioned the merits of mass treatment . what appears to be an unwillingness or an incapacity to engage with local concerns of these kinds , and specifically to address matters such as pluralistic approaches to aetiology and treatment in health education materials and training of distributors , is by no means unique to staff working for the national programme to control neglected tropical diseases in tanzania ( see , for example , research on the control of schistosomiasis and soil - transmitted helminths in uganda by parker et al . ( 2008 ) and parker & allen ( 2011 ) , and research on malaria by hausmann muela et al . in tanzania ( 1998 , 2002 ) ) . it does , however , have unfortunate consequences as it ends up confirming , rather than refuting , existing outlooks and fuels anxieties about the merits of swallowing drugs provided by international donors and distributed through government channels . that said , it would be a great mistake to assume that more extensive and sophisticated communication will be sufficient to improve drug uptake . the current system of rolling out free drugs relies upon voluntary labour to run the programme and this , too , is problematic . village health workers , otherwise known as the community 's own resource persons ( corps ) take responsibility for distributing drugs for the treatment of mabusha and matende ( malecela et al . , 2009b ) . that is , health workers at a national level train health workers at a regional level ; regional staff train staff working at a district level ; and district staff train those working at a ward and village level ( malecela et al . , 2008 ) . village drug distributors are not paid for the time they spend handing out drugs within their villages , but they receive a small sitting allowance for attending training sessions . while positive statements have been made about the effectiveness and efficiency of the cascade system ( malecela et al . , 2008 , 2009b ) , observations and discussions with health workers and other villagers suggest that the current system does not always run smoothly . in particular , it overlooks the fact that some villages cover significant geographical areas and there are often significant constraints preventing distributors from taking the time to contact those living on the outer reaches of their village . to walk from home to home , persuading individuals with whom there is little social connection , requires a big commitment . this is expected to occur in addition to a normal day 's work , and may need to be done repeatedly , because many individuals may be absent , perhaps fishing or farming or visiting relatives . crucially , the reliance upon a small number of adults to distribute drugs within each village also assumes that a sense of goodwill and concern extends to all residents equally . in many places , this is not the case . in mwembeni , for example , the makonde are , in social and political terms , much more marginal than their muslim neighbours . they are often referred to as kafir . as a result , many of them are not even recorded in the village register . in addition , it became apparent that by 2010 a parallel system of drug distribution had emerged . that is , two muslims were taking responsibility for distributing drugs throughout the village . christian makonde living reasonably close to the centre of the village were told to come to a central location to collect drugs , but those living far from the centre were not informed of the treatment . a 55-year - old makonde man , living on the outskirts of the village , summed up the situation when he revealed that he had never heard about the free provision of drugs as : the only information we get is when we have to contribute something the other news is not delivered. by contrast , many of the friends , neighbours and relatives of the drug distributors were being given drugs in their own houses . somewhat similar issues were also noted in kigombe , where divisions between political parties , between muslim factions and between muslims and christians all affected distribution activities . as in mwembeni moreover , in 2007 , one of the imams was reported to have openly opposed distribution activities to his followers , and the distributors lacked the authority or knowledge to challenge him . such diverse ( and often very locally specific ) divisions in what are perceived externally as homogeneous communities obviously affect drug distribution . indeed , close scrutiny of a programme like tanzania 's national lymphatic filariasis elimination programme makes manifest social hierarchies and fissures . they become particularly clear in ethnographic research , though they may be hard to enumerate . although efforts were made to see if it was possible to discern consistent patterns of treatment exclusion in the data that were collected during semi - structured interviews on drug uptake , this did not prove particularly insightful . political affiliation , for example , was too sensitive an issue to be asked in all interviews , and it was sometimes difficult to assess the accuracy of answers when they were forthcoming . systematic effects of the manifest conflicts and tensions in the muslim population were similarly hard to gauge and quantify . however , with respect to drug distributers coverage of resident christians , it was possible to make a basic assessment in mwembeni . here , there were some christians living near the primary school , not far from the village centre , or who had children at the school . it was those residing at the periphery of the village that were omitted . also , a general bias towards offering tablets to muslims in preference to christians was discernible in the self - reported uptake data : between 2004 and 2007 , 53% of muslim adults reported taking the tablets in mwembeni , and 48% of christians . ethnographic research on this and other issues revealed that there were marked differences of attitude and commitment among drug distributors . observations and discussions strongly indicated that it was the most marginal that end up being excluded from the offer of treatment . it remains to be explained why self - reported drug uptake is higher in some places than others . it relates to levels of engagement with the target populations and resulting increased capacity of people to make informed choices . it has already been observed that the proximity of the official sentinel site near tanga town at kirare and the visits of medical staff and researchers explain a relatively high self - reported uptake rate , even if it is less than is necessary for long - term control or elimination of the disease . similar rates were noted in this study , in bwembwera and magila in muheza district and at the small trading centre of jaira in pangani district . the case of jaira was discussed with district medical staff based at the hospital in pangani town in 2011 . the district medical officer commented that the improvement in self - reported drug take - up ( from a low of 40% in 2007 to around 70% in 2010 ) must have been to do with the direct involvement of his staff . some of his staff had been to jaira at the time of distribution and had responded personally to the questions and concerns of the population . it was not possible to do so elsewhere due to lack of transport and lack of qualified personnel who might be deployed . with respect to bwembwera and magila , the take - up rates of 68% in 2006 and 65% in 2007 in bwembwera , and 70% in 2007 in magila , probably reflect their proximity to muheza town and to muheza hospital . it was found that in these locations knowledge about lymphatic filariasis and the rationale for mass treatment had been more adequately communicated than in locations near the coast . in addition , it was reported by informants that surgery had been made available to those men seeking it through an established system of referral , rather than the kind of ad hoc surgery camp that was run in pangani in 2008 . district medical staff additionally noted that higher rates of drug uptake near to muheza town reflected the better training and larger number of drug distributors than was the case in more remote locations . they were well aware of the difference in levels of engagement within the district , and explained that they just did not have the resources to run a more effective operation at a distance from their base . the research presented in this article is based upon fieldwork undertaken in northern coastal tanzania . it demonstrates that the uptake of drugs at selected study sites in pangani and muheza districts falls short of the sustained high coverage levels required to eliminate lymphatic filariasis , and the prospect of eliminating the disease by 2020 with current strategies is remote . in broad terms , the findings presented in this article add to the worrying conclusions of simonsen et al . ( 2010 ) , who report that drug uptake at the nearby and closely studied sentinel surveillance site at kirare , while higher than those reported at the study sites in this paper , is insufficient ; and that mda is having declining effects on disease control . in combination with research undertaken by michael et al . ( 2010 ) , results presented here highlight the pressing need to fine - tune mass treatment programmes according to ecological , biological and social factors operating at a local level . the results also highlight the need for more adequate monitoring , recognizing that there are risks involved in sustaining mda with partial coverage over time , including risks associated with drug resistance ( mccarthy , 2005 ) . elsewhere in the country considerable success , for example , has been reported in zanzibar ( mohammed et al . the prevalence of microfilariae has fallen from 17.8% and 7.2% to less than 2% at two sentinel surveillance sites , following five annual rounds of mass treatment . where there are apparent successes of this kind , it would be helpful to know exactly why they have occurred . the present trend in the ntd literature , driven by target - orientated funding , is to assume they are inevitable . explanations for the reported achievements in zanzibar include sustained high mda coverage of 7080% over a five - year period , linked to the introduction of effective lymphoedema management programmes . it is probably also significant that the prevalence of microfilariae was initially lower than that recorded at kirare . it is not clear , however , whether these lower levels were due to other vector control activities that may have been running in the vicinity for lymphatic filariasis and/or malaria or whether they reflect the relative affluence and ease with which zanzibari populations can access public health interventions and other forms of biomedical care compared with populations in tanga region on the mainland . whatever the explanation(s ) , it is dangerous to simply assume that the results can be readily replicated . there are specific reasons why mda works in some places and fails in others ; and it is crucially important to know what these reasons are . the mixed effects of mda for lymphatic filariasis have specific aspects but in other ways are not unique . mass distribution of drugs for other neglected tropical diseases faces comparable problems . in tanzania , hastings documented the uptake of ivermectin and albendazole for the treatment and prevention of onchocerciasis in doma , morogoro region , in 2007 . working with a 20% sample involving 150 randomly selected adults , her research suggested that uptake fell from 31% in 2004 to 29% in 2006 and 17% in 2007 ( hastings , personal communication ) . wingate - saul documented the uptake of the drug praziquantel for the treatment and prevention of infection with s. mansoni . working in fourteen villages in ukerewe island , her research suggested that in 2007 drug uptake among adults ranged from 18% to 74% , with the average uptake being 44% ( wingate - saul , 2008 ) . these rates are well below target levels , and it is not clear what effect they are having on the prevalence of infection . meanwhile , a disturbing indicator of how local fears and rumours can spiral out of control are the violent riots that occurred in several locations during 2008 in response to mda for the treatment and prevention of s. haematobium in schools . hastings had to be rescued from her research location near morogoro by armed tanzanian police . some teachers found distributing drugs were not so lucky and were beaten by angry mobs convinced that tablet distribution had sinister motives , such as population control ( irin humanitarian news and analysis , 2008 ; daily news online edition , 2008 ) . there are , of course , well documented accounts of social resistance to other top - down biomedical disease control programmes such as vaccination programmes to control polio in nigeria ( yahya , 2010 ; renne , 2011 ) and tetanus in cameroon ( feldman - savelsberg et al . , 2000 ) ; as well as the provision of condoms to prevent the transmission of hiv / aids in parts of indonesia ( butt , 2005 ) , botswana ( heald , 2000 ) and south africa ( niehaus & jonsson , 2005 ) . with respect to neglected tropical diseases , there has also been some active resistance to treatment in uganda , if not on the scale of the tanzanian riots . these have been described in relation to mda for schistosomiasis in north - west uganda ( parker et al . , 2008 , 2012 ; how , then , should fears associated with the mass distribution of drugs for the treatment and prevention of lymphatic filariasis be addressed ? it may be tempting for policymakers and planners to feel that the most effective way to respond to the low uptake of drugs for lymphatic filariasis is to continue to focus on narrow technical and logistical aspects of drug delivery as there is little they can do to counter rumours that the government and international agencies have hidden agendas that do not serve the interests of local people . however misplaced local fears and anxieties about free treatment actually are , they are also ideas that are widely held and have emerged from experiences and empirical observations . white ( 2000 ) , reflecting on the persistence of vampire stories in africa , and geissler & pool ( 2006 ) , analysing rumours associated with medical research projects in sub - saharan africa , have demonstrated the multiple ways in which stories and rumours articulate local understandings of the workings of power and knowledge . given that it is politically and economically neglected people who are mostly infected with neglected tropical diseases , it is hardly surprising that the targets of vertical mda projects do not necessarily take the stated intentions of those running them at face value . there is a need for a real engagement with such populations , necessitating different kinds of local - level strategies . with respect to lymphatic filariasis a range of interventions are required , and social empowerment needs to be part of the mix . new ways have to be found to explain the rationale for mass drug administration ; that mabusha and matende are signs of the same infection ; that it is possible to be asymptomatic and infected with parasitic worms ; that the drugs being provided will not necessarily reduce the swelling of those suffering from matende or mabusha ; that the purpose of combining the drugs invermectin and albendazole is to prevent those who are infected but asymptomatic from developing clinical signs of infection ; and that the drug albendazole also has the capacity to treat those suffering from other worms , notably soil - transmitted helminths . there has to be knowledge transfer for afflicted people to be able to make informed choices . as allotey et al . ( 2010 ) note , the current emphasis on context - free , pathogen - focused ntd control is a return to magic bullet medicine , and ignores the fact that vaccines and drugs do not cure neglect or poverty . perhaps the largest challenge of all is not how to win round the hearts and minds of those being targeted for treatment , but rather how to create a space whereby those engaged in developing policies at a global level feel able to acknowledge that the types of difficulties discussed in this paper are significant and that raising them is helpful and relevant for developing more effective strategies . considerable institutional pressures mitigate against effective action , not least because intense competition for funding discourages critical thinking and analysis ( allen & parker , 2011 , 2012 ) . there is a tendency towards disingenuous dismissal of important information , both social and biological , and an overemphasis on quickly achieving optimistic goals . it has led to some questionable claims about what has already been achieved , and to some strongly worded responses to the kind of research presented here . a global commitment to control or eliminate lymphatic filariasis and other neglected tropical diseases is a hugely positive development , but that is all the more reason to avoid treating fundraising rhetoric as facts , and to learn lessons from what has happened with such large - scale vertical schemes in the past . as a 2010 editorial in the lancet observed : currently , it is only an afterthought without proper monitoring and accountability , countries and donors and taxpayers have no idea whether or how their investments are working, ( the lancet 2010 , p. 526 . ) with respect to mda , there is a need for sober and rigorous assessment , which examines locally specific effects and subjects reported results to appropriate critical scrutiny . even if claims about making poverty history and alleviating the suffering of the bottom billion are set aside as overblown , it is certainly possible that programmes have potential to assist some poor and marginal populations . however , without heeding warnings and amending strategies , that window of opportunity may be lost .

summarythis article documents understandings and responses to mass drug administration ( mda ) for the treatment and prevention of lymphatic filariasis among adults and children in northern coastal tanzania from 2004 to 2011 . assessment of village - level distribution registers , combined with self - reported drug uptake surveys of adults , participant observation and interviews , revealed that at study sites in pangani and muheza districts the uptake of drugs was persistently low . the majority of people living at these highly endemic locations either did not receive or actively rejected free treatment . a combination of social , economic and political reasons explain the low uptake of drugs . these include a fear of treatment ( attributable , in part , to a lack of trust in international aid and a questioning of the motives behind the distribution ) ; divergence between biomedical and local understandings of lymphatic filariasis ; and limited and ineffective communication about the rationale for mass treatment . other contributory factors are the reliance upon volunteers for distribution within villages and , in some locations , strained relationships between different groups of people within villages as well as between local leaders and government officials . the article also highlights a disjuncture between self - reported uptake of drugs by adults at a village level and the higher uptake of drugs recorded in official reports . the latter informs claims that elimination will be a possibility by 2020 . this gives voice to a broader problem : there is considerable pressure for those implementing mda to report positive results . the very real challenges of making mda work are pushed to one side adding to a rhetoric of success at the expense of engaging with local realities . it is vital to address the kind of issues raised in this article if current attempts to eliminate lymphatic filariasis in mainland coastal tanzania are to achieve their goal .

Introduction Previous research on lymphatic filariasis in mainland coastal Tanzania Field sites and methods Uptake of drugs for the treatment and prevention of lymphatic filariasis in Pangani and Muheza districts, Tanga region Why is the uptake of drugs for the treatment and prevention of lymphatic filariasis low? Conclusion

PMC3982338

infectious adenitis has been rarely reported in neonates , 40 cases of neonatal suppurative parotitis and only 17 cases of isolated submandibular sialadenitis have been reported in english literature . we describe a 18 days old neonate with suppurative parotitis associated with submandibular abscess , which was treated by incision and drainage . the present case report is about an 18 days old full term female neonate presented to our department with swelling in parotid and submandibular area on left side for 6 days . the local examination revealed a swelling about 5 cm 4 cm in size in parotid area extending down to submandibular and submental area . overlying skin was erythematous and swelling was fluctuant [ figure 1 ] . left parotid and submandibular abscess further examination revealed that upon pressing the swelling , purulent material was extruding through the floor of the mouth [ figure 2 ] . the base line investigation were done which showed leucocytosis - 18,000/l with neutrophillis - 80% , kidney and liver function tests were normal , incision and drainage of the abscess was done about 30 - 40 ml of frank pus was drained . patient was already put on amoxicillin + clavulanate ( 15 mg / kg orally every 12 h ) . after two weeks and then at 1 month of discharge from hospital , baby had completely recovered . suppurative parotitis is usually unilateral and may progress to abscess formation . among salivary glands ; the involvement of parotid gland is more common than submandibular gland . . the risk of parotitis in premature babies may be related to dehydration leading to stasis in the salivary gland ducts due to reduced secretions . most common mode of spread is tracking of oral flora in a retrograde fashion into gland , although gland may get invaded with bacteria through the blood stream . decreased salivary production and stasis , dilatation of ducts through scaring or obstruction by stone , variation in ductal structure may lead to retrograde flow from oral cavity . another potential cause of transmission of bacteria is during the breast feeding and a through contaminated formula , as in the present case infant was breast fed , but the mother had no signs of mastitis , as the history of maternal mastitis can act as a source of bacterial invasion as described by tapsz et al . breast feeding can not be considered as a risk factor for suppurative parotitis as there are more chances of contamination with formula feeds . as in our case the history of initial appearance of swelling and erythema were in the preauricular area which then progressed subsequently to involve the submandibular and submental area . examination with ultrasound is readily available , cheap , noninvasive and useful for diagnosis and differential diagnosis and excluding abnormality of stenosis of the duct , siolith . purulent drainage from stensen 's duct is diagnostic of the suppurative parotitis as is demonstrated in our case . in a report of spigel et al . , the most common pathogen was s. aureus , which was found in 55% of the patients . in our patient , the main stay of the treatment is to use antibiotics to cover the causative organism i.e. , s. aureus . however as our baby had large fluctuant swelling we decided to do the incision and drainage of the abscess . we found that after incision and drainage antibiotics had to be given for 1 - 2 days and the hospital stay was also reduced by 4 - 5 days . although incision and drainage is less frequently used in this modern antibiotics era but it has still got a very important role in frank abscess formation as in our case . incision and drainage also helps to isolate the organism and appropriate antibiotic needed in case of some residual purulent material remains in the abscess cavity . although the parotitis is very rare in neonates as described it should be suspected in neonates with erythematous preauricular swelling . delay in diagnosis and treatment may lead to involvement of surrounding glands as in our case . and in case of frank purulent nature of the abscess treatment should be incision and drainage which the promptly decreases the burden of septic focus , duration of antibiotic therapy and hospital admission .

suppurative involvement of salivary gland in neonates is a rare disorder . parotid gland being the most commonly involved . we described a case of suppurative parotitis leading to abscess formation and subsequent involvement of the submandibular gland . incision and drainage of the abscess was performed , most of the purulent material was drained . symptoms and signs resolved within 2 days . pus culture grew staphylococcus aureus

INTRODUCTION CASE REPORT DISCUSSION

PMC3015436

a curriculum consisting of short practice sessions would help experienced surgeons and surgical residents maintain and refine essential surgical skills . several steps , though , are required before such a program can be established . first , the essential surgical skills common to all specialties must be identified and then the physical restraints that influence the teaching of these basic skills must be noted . this is essential for determining how many lesson plan units will be required . regardless of the skill being taught , accompanying text , drawings , or video clips will be needed for explanation purposes . one lesson plan unit consists of either of these visual aids , or a combination of them . essential surgical skills must be defined as either basic skills or advanced skills , with the latter using the former as a foundation . for example , the placement of a simple suture involves practice first using a laparoscopic needle holder to engage different types of needles . next , needle advancement must be achieved through the two tissue borders to be approximated . once these basic skills are mastered , the surgeon can then perform advanced skills such as interrupted or continuous anastomoses on different organ structures . physical restraints also have a bearing on teaching essential surgical skills . in open surgery , once proper isolation of the operative field is achieved , hand movements proceed easily . in laparoscopy , exposure is excellent , but the surgeon 's movements are labored . patient and surgeon body habitus , port placement , instrument length , and instrument handles can present major obstacles during laparoscopy . if all essential surgical skills and their affecting physical restraints are identified and taken into consideration , can an all - inclusive curriculum be developed ? although some disagreement might exist as to the order of importance in teaching essential surgical skills , the following order is suggested : 1 ) knot tying ; 2 ) simple interrupted suturing ; 3 ) simple running suturing ; 4 ) purse string suturing ; 5 ) end - to - end anastomosis ; 6 ) end - to - side anastomosis ; 7 ) side - to - side anastomosis ; 8) use of stapling devices ; 9 ) catheterization of ductal structures . the second subset involves parameters that will depend on the essential surgical skill being practiced on at the time . five general physical restraints exist ( table 1 ) that were never encountered in open surgery but that are always present in laparoscopic surgery . they are considered inherent to the very nature of the technology and present unique challenges to surgeons . understanding them is essential in trying to develop teaching models as well as in creating new instruments . excessive instrument length variations in tissue plane angles excessive instrument length is a new surgical parameter unique to laparoscopy and the possible cause of fatigue and injury to a surgeon 's upper extremities . in contrast to open surgery , where the surgeon 's hands are almost always hovering over the operative site at convenient angles , in laparoscopy limited port position restricts the surgeon 's hands to distant and narrow working angles . what is won as a benefit in video surgery with increased magnification and exposure is lost in the lack of flexibility of motion and increased fatigue . because the surgeon 's hands are closer to the operative site in open surgery , less energy is used in moving the instrument working jaws . this is in direct contrast to laparoscopic surgery where the hands are a considerable distance from the operative site , requiring a greater expenditure of energy to perform the same maneuvers . imagine that you have two people on a record turntable starting at position a and b , a being at a greater distance from the center c than b. for both to maintain the same amount of rotations per minute , which will result in a going to a ' and b going to b ' , it will require a to travel a longer distance at a faster velocity and greater energy expenditure , than it will for b. the physics involved in this model is directly applicable to the physics of laparoscopy . as a result travel around a fixed point c. arc distance aa ' is greater than bb ' . rotational effects seriously hamper training efforts and are related to the standing position of the surgeon in relation to the camera view projected on the monitor . it is not enough to practice skills , such as suturing and knot tying , in the camera position alone , but also in the right camera position , left camera position , and the opposite camera position . this is essential because the movements necessary to tie a knot in one position are entirely different from those needed to tie the same knot in another . to practice advanced skills in only the camera position and to forget the other positions is impractical when performing videoscopic surgery that requires the use of assistants to help retract and assist in important surgical maneuvers . everyone can not be located at the camera position when only one camera is being used . variations in tissue plane angles are evident because of the cavernous nature of the operative field in videoscopic surgery . for example , when performing a laparoscopic bladder neck suspension procedure , placing a simple suture in the periurethral tissues requires a different set of maneuvers than placing the same suture on cooper 's ligament , which is approximately 90 degrees up the pelvic sidewall . a repair of an anterior abdominal wall ventral hernia might conceivably require placing sutures while aiming up to the ceiling from the site of entry ports . a simulation model must provide practice sessions in all the possible varying tissue plane angles that the videoscopic surgeon might encounter in actual practice . the first is , the tissue angle , which is defined as that angle between the tissue plane being worked on and the patient 's horizontal axis . the second is , the instrument angle , which is the angle between the shaft of the laparoscopic instrument and the patient 's vertical axis . a cylindrical drawing is used to represent tubular structures , such as vessels , ducts , or bowel . : tissue angle , : instrument angle , tp : tissue plane , ip : instrument plane , s : tubular specimen , a : diaphragm , b : instrument shaft , c : entry port , d : abdominal wall , e : pelvic side wall , x : patient horizontal axis , y : patient vertical axis . representation of several tissue angles along the abdominal cavity . although variations may exist among the angles of organs in individuals , for the most part they will be similar . the ideal angles to provide maximum flexibility and minimum surgeon fatigue are yet to be worked out . to simulate variations in tissue plane angles , a special device to hold practice specimens is needed . it must permit placing all types of specimens in as many angles from the horizon as possible . specimen orientation has to do with how , for instance , a tubular structure , such as a bowel or a vessel , lies on the particular plane it is on . the tissue plane , at angle , is demonstrated as tp extending to t'p ' along the patient 's third dimensional axis z. within the two - dimensional plane , three tubular structures , a , b , and c , are placed . in relationship to the surgeon 's point of view on the monitor , they lie at either a horizontal , vertical , or oblique position . specimen orientation at tissue angle a. a : horizontal , b : vertical c : oblique . x : patient horizontal axis , y : patient vertical axis , z : 3rd dimensional axis perpendicular to x & y. in open surgery , if tissue rotation is off , the surgeon instinctively rearranges his field or himself to provide better exposure . unfortunately , this luxury is not always available in laparoscopy , and the surgeon must learn to work in undesirable orientations to do the job . depth perception errors arise from the fact that we work in three - dimensional space , but when performing videoscopic surgery , the monitor image is a two - dimensional replication of the operative field . so , if we are tying a suture using a monitor display , we often run into the problem of determining how close up or far away a suture end is from the rest of the suture length we are working with . it requires practice to observe important landmarks to determine what part of a suture lies in front and what part lies immediately behind adjacent structures before knot tying can be performed effortlessly . in addition to the five general physical restraints mentioned above , various other particular physical restraints exist ( table 2 ) that come into consideration when developing a video training program . these are important factors that affect the number of lesson plan units to be developed and reflect the personal preferences of both the student and the teacher . just as in open surgery , we all have our own particular way of performing a series of actions so too in videoscopic surgery these differences also exist . this is not a matter of a right or wrong way of doing things , but merely represents the fact that there is more than one way to accomplish a goal . needles shape : curved or straight suture type : braided or monofilament suture size : large or small suture material : soft or stiff suture designs : straight or looped specimen type : organic meats or synthetic models handedness of surgeon : right - handed or left - handed surgical approach : intracorporeal or extracorporeal type of pelvic trainer : closed box , ring , computer simulation needle shape is important in facilitating entry into tissue planes and in interacting with the needle holder . the size of the needle also has an impact on the ease or difficulty encountered in manipulating it . the type of suture used and the suture size will depend on the tissue being worked on . tying ease will vary in different situations . the suture design can vary from a classic straight suture or one with loops built in to facilitate handling and eliminate some of the motions of tying . the handedness of the surgeon must be taken into consideration as well because the hand movements of each will be completely different , requiring additional specific drawings or video clips , depending on whether the surgeon is right - handed or left - handed . surgical approach concerns the ability to tie a knot either completely intracorporeally with instruments versus assisted extracorporeal hand directed suture tying . in the latter , some form of pusher goes in and out of one of the ports to tighten the knot . they are made of either clear or opaque materials that may permit the surgeon to see the practice field . the expense associated with maintaining their function unfortunately is far beyond what most hospitals or solitary practitioners can afford . their main importance appears to lie in the computer technology that allows a surgeon to operate by means of robotics from a distant location . a major concern about this technology is what happens when a mechanical malfunction occurs in the middle of a critical operation , and the only persons in the room , besides the patient , are an anesthesiologist and a mechanic . who is going to stop the bleeding then ? using some of the variables involved in both the general and particular physical restraints as a guide , let us see how many lesson plan units would be needed to do a thorough job of teaching how to tie a vessel intracorpo - really . a fairly inclusive approach would take into consideration the following parameters : ( 2 ) type of pelvic trainers : opaque box and ring(4 ) standing position : camera , right camera , left camera , and opposite camera(3 ) tissue plane angle : 0 , 30 , and 90(3 ) specimen orientations : horizontal , vertical , and oblique(2 ) specimen model : artificial and animal tissue(2 ) suture material : polyfilament or monofilament(2 ) suture size : large : 0 and small : 4 - 0(2 ) suture design : straight and looped(2 ) handedness : right and left ( 2 ) type of pelvic trainers : opaque box and ring ( 4 ) standing position : camera , right camera , left camera , and opposite camera ( 3 ) tissue plane angle : 0 , 30 , and 90 ( 3 ) specimen orientations : horizontal , vertical , and oblique ( 2 ) specimen model : artificial and animal tissue ( 2 ) suture material : polyfilament or monofilament ( 2 ) suture size : large : 0 and small : 4 - 0 ( 2 ) suture design : straight and looped ( 2 ) handedness : right and left to obtain the total number of lesson plan units needed to do a fairly thorough job of teaching the essential surgical skill of tying off a vessel , we need to multiply the above numbers in parenthesis together . this is the total number of lesson plan units needed to do a reasonably thorough job of teaching how to tie a vessel , while taking into consideration the aforementioned general and physical restraints . although this number appears large , it still does not include all the variables of individual preferences that might still exist . a more limiting approach would be to restrict the parameters as follows : ( 1 ) type of pelvic trainer : ring(1 ) standing position : camera(1 ) tissue plane angle : 30(2 ) specimen orientations : horizontal and vertical(1 ) specimen model : artificial(1 ) suture material : silk(1 ) suture size : large : 2 - 0(1 ) suture design : looped(1 ) handedness : right ( 1 ) type of pelvic trainer : ring ( 1 ) standing position : camera ( 1 ) tissue plane angle : 30 ( 2 ) specimen orientations : horizontal and vertical ( 1 ) specimen model : artificial ( 1 ) suture material : silk ( 1 ) suture size : large : 2 - 0 ( 1 ) suture design : looped ( 1 ) handedness : right by limiting training sessions to only the above physical restraints , our total number of lesson plan units now comes down to 1x1x1x2x1x1x1x1x1 = 2 . these two lesson plan units would consist of video clips or still photographs accompanied by descriptive text . the description of these two lesson plan units is as follows : lesson plan unit 1 demonstrates a right - handed surgeon using a ring simulation device to practice vessel tying by an intracorporeal technique with a 2 - 0 silk looped suture while standing in the camera position . the artificial vessel specimen is at a 30 inclination and lying in a horizontal orientation . lesson plan unit 2 demonstrates a right - handed surgeon using a ring simulation device practicing vessel tying by an intracorporeal technique with a 2 - 0 silk looped suture while standing in the camera position . the artificial vessel specimen is at a 30 inclination and lying in a vertical orientation . the previous analysis leads us to believe that an all - inclusive curriculum can never be developed . if teaching one essential surgical skill thoroughly requires over 2,000 lesson plan units , then to teach even 5 would result in the need for 10,000 . to be completely thorough , the course would take years to develop and years for surgeons to perform all the lessons . by necessity to maintain reasonable time and monetary schedules , training courses will always have to be limited arenas for surgeons to practice in . the fact that an entirely all - inclusive laparoscopic training course can not be developed does not mean we should abandon the concept . what course directors must continue to do is concentrate on those areas of expertise they are most familiar with while remaining open to the fact that other ways may exist for performing similar techniques . the fact is that whatever is being covered in any one course may actually be just a small part of the knowledge that is still to be uncovered . because laparoscopic surgery training is such a paradox , we must constantly be on guard against establishing this technology is still in its infancy , and its final chapter is unwritten . if we try to standardize operations , we will freeze progress and inhibit surgeons from attempting new and far better approaches to current laparoscopic procedures . what is needed is to add to the current excellent clinical training courses available , a method that allows surgeons to practice essential surgical skills in well - defined limited lesson plan units using an inexpensive laparoscopic simulation device . by this means , surgeons can then take the initiative for themselves to further fine tune course - acquired skills preferably in a trainer that allows variation of as many of the previously mentioned physical parameters as possible .

despite the acceptance of laparoscopy for performing routine operations , a need still exists for experienced surgeons and surgical residents to maintain and refine essential surgical skills . unless used on a frequent basis , laparoscopic skills are not easily maintained . in addition , when new laparoscopic instruments are introduced , surgeons need a way to practice using them that does not involve immediate patient contact . novice surgeons need the most training of all and ideally would be best served using a standardized teaching curriculum that would cover as many of the basic laparoscopic parameters as possible . this article discusses how best to set up a laparoscopic simulation training program that covers as much ground as necessary , while respecting the restraints of time limitations and monetary concerns

INTRODUCTION STUDY OBJECTIVES MEANS AND METHODS RESULTS CONCLUSION DISCUSSION

PMC4863118

sepsis is still a leading cause of death among critical patients in the intensive care units , and the life quality of the survivors would usually be impaired [ 15 ] . there was a serious decrease of immunocytes , including b / t - lymphocytes , dendritic cells ( dcs ) , gastrointestinal epithelial cells , and even thymocytes , at the beginning of sepsis as shown in both animal models and septic patients [ 69 ] . it has been noted that the septic patients would gradually enter immunosuppression after primary hyperinflammatory response , which is defined as immunoparalysis [ 2 , 4 , 6 , 7 ] . in recent years , investigators have become interested in the study of the mechanisms regarding immunosuppression and the development of new methods to regulate immune response during sepsis , including both activation of tregs and apoptotic depletion of immunocytes . as a class of cd4 t cell subsets , tregs are a group of specialized immune cells that play an important role in immune homeostasis . during the development of sepsis , tregs subdue inflammation and tissue damage , and they may also cause immune dysfunction , such as induction of t - lymphocytic apoptosis , inhibition of cd4/cd8 t - lymphocytic function , and mediation of shifting from the helper t cell 1 ( th1 ) to th2 response , especially immunoparalysis via expression of ctla-4 and tgf- , as well as anti - inflammatory cytokines ( il-10 and tgf- ) [ 1217 ] . nrp-1 is characterized as a single - pass transmembrane glycoprotein , which is originally described to be involved in axon guidance , revascularization , and tumorous growth [ 18 , 19 ] . recently , nrp-1 , an essential component of the immunological response in humans and animals , is identified as a potent surface marker for cd4cd25tregs [ 2023 ] . in addition , the expression of nrp-1 on tregs was correlated with the expression of foxp-3 and suppressive capacity . solomon and his colleagues further showed that nrp-1cd4cd25tregs suppressed the proliferation of cd4cd25 t cell more efficiently than cd4cd25tregs and nrp-1cd4cd25tregs in a mouse model of experimental autoimmune encephalomyelitis ( eae ) . in our previous study , we indicated that tuftsin - derived t - peptide , the typical ligand of nrp-1 , had the ability to improve the outcome of septic mice in a dose- and time - dependent manner and was associated with downregulation of the negative immunoregulation of tregs and improvement of the microenvironment of cellular immunosuppression in septic mice . thus , further investigation on the impact of nrp-1 on the negative immunoregulation of tregs will provide a new target for the study of immune regulation in sepsis . in the present study , the objective , using the classical septic model , that is , clp , is to investigate the impact of sepsis on the expression of nrp-1 on cd4cd25tregs and the negative immunoregulation of nrp-1cd4cd25tregs in septic mice , as well as the potential therapeutic value of nrp-1 in sepsis . we demonstrated that the expression of nrp-1 on cd4cd25tregs was significantly upregulated in a grade- and time - dependent manner , nrp-1cd4cd25tregs possessed primary negative immunoregulation in septic response , and recombinant nrp-1 polyclonal antibody markedly downregulated the demethylation of foxp-3-tsdr in the stimulation of lps in a dose - dependent manner . nrp-1 could represent a new potential therapeutic target for the study of immune regulation in sepsis . inbred male balb / c mice ( laboratory animal center of chinese academy of medical sciences , number scxk - jing-2009 - 0007 , beijing , china ) , aged 68 weeks , weighing 20 2 g , were used in the present study . all procedures were undertaken in accordance with the criteria of national institute of health guide for the care and use of laboratory animal and approved by the scientific investigation board , medical college of chinese pla , beijing , china . the medium used throughout the in vitro experiment was rpmi1640 ( containing 100 u / ml penicillin , 100 l / ml streptomycin , and 1.5 mm glutamine ) with 10% heat - inactivated fetal bovine serum ( fbs ) . cd4cd25 regulatory t cell isolation kits , goat anti - rabbit igg microbeads , rabbit anti - mouse nrp-1 , and ld / sm columns were purchased from miltenyi biotec gmbh , bergisch gladbach , germany . fluorescein isothiocyanate- ( fitc- ) conjugated annexin - v apoptotic kit was purchased from nanjing keygen biotech , nanjing , china . purified hamster anti - mouse cd3e and cd28 were purchased from bd pharmingen , san diego , ca . antibodies used for flow cytometric analysis , including fitc - conjugated anti - mouse / rat - foxp-3 , fitc - conjugated anti - mouse cd152 ( ctla-4 ) , and allophycocyanin- ( apc- ) conjugated anti - mouse / rat - tgf- , were purchased from ebioscience , san diego , ca . apc - conjugated anti - mouse / rat - nrp-1 and recombinant nrp-1 polyclonal antibody were purchased from r&d systems , minneapolis , mn . enzyme - linked immunosorbent assay ( elisa ) kits for interferon- ( ifn- ) , il-2 , il-4 , il-10 , and tgf- were purchased from excell biol , shanghai , china . ketamine and su - mianxin - ii ( containing 2,4-xylazole , ethylenediaminetetraacetic acid , dihydroetopine , and haloperidol ) were purchased from china academy of military medical sciences , beijing , china , and they were used as the anesthesia for animals . spleens were harvested and prepared into single cell suspension by passing through a 30 m stainless steel mesh twice and then treated with ficoll - paque density gradient centrifugation . cd4cd25tregs and cd4cd25 t cells were isolated from mononuclear cells using mouse cd4cd25treg isolation kit and minimacs separator ( miltenyi biotec gmbh , bergisch gladbach , germany ) according to manufacturer 's instructions . cd4cd25tregs were incubated with a rabbit anti - mouse nrp-1 antibody ( abcam , cambridge , ma ) for 20 minutes at 4c , washed and incubated with goat anti - rabbit igg microbeads for 30 minutes at 4c , and selected for nrp-1cd4cd25tregs and nrp-1cd4cd25tregs by minimacs separator according to manufacturer 's instructions . isolated cells were cultured in rpmi 1640 supplemented with 10% fcs . after being anesthetized , a 0.5 cm incision was made on the abdomen of mouse , and the cecum was exposed . the cecum at the designated position between its distal pole and ileocecal junction was ligated for the desirable degree of sepsis : 1/3 for low - grade sepsis , 2/3 for midgrade sepsis , and ligated ileocecal junction for high - grade sepsis . the diameter of puncture needle was 0.6 mm , and it was used to induce clp in the experiment . the mice were given a subcutaneous injection of 0.9% sterile saline solution with an amount of 40 ml / kg body weight after clp . 150 mice were used to investigate the severity - dependent response between the expressions of nrp-1 and foxp-3 of tregs , and they were divided into five groups : control group , sham group , and three different clp groups ( low - grade , midgrade , and high - grade ) , with 30 mice in each group . with the optimal degree of sepsis , another 150 mice were employed to observe the time - dependent response between the expressions of nrp-1 and foxp-3 of tregs , and they were divided into five groups : control group and clp with four interval groups ( 12 , 24 , 48 , and 72 hours ) , with 30 mice in each group . the survival time and rate of various groups the optimal grade and time point were used to investigate the impact of nrp-1 on the negative immunoregulation of tregs in sepsis . they were divided into four groups with the same cell number : control group and clp with three different subtype tregs ( cd4cd25tregs , nrp-1cd4cd25tregs , and nrp-1cd4cd25tregs ) . they were cocultured with conventional cd4cd25 t cells for 24 hours in a ratio of 1 : 1 and treated with anti - cd3 ( 5 g / ml ) and anti - cd28 ( 2 g / ml ) antibody for polyclonal activation of t cells , respectively [ 1416 ] . the proliferative activity , apoptotic rate , and secretive ability ( including interferon- ( ifn- ) and il-4 ) of cd4cd25 t cells and the expression of foxp-3/ctla-4/tgf-/nrp-1 , apoptotic rate , and secretive ability ( including il-10 and tgf- ) of every subtype of tregs were determined . in vitro , nrp-1cd4cd25tregs were isolated from normal mice spleen and cultured with different doses ( including 1 , 10 , 100 , 1000 , and 10000 g / ml ) of recombinant nrp-1 polyclonal antibody in the stimulated lps ( 1000 ng / ml ) for 24 hours . the demethylation level of foxp-3-tsdr was determined by demethylation - sensitive rt - pcr , which had been recounted by tatura et al . . the proliferative activity of cd4cd25 t cells was determined by cck-8 according to protocols provided by the manufacturer . the absorbance was read in microplate reader ( spectra mr , dynex , richfield , mn ) at od 450 nm . cd4cd25tregs were stained with fitc - conjugated anti - mouse - ctla-4 , apc - conjugated anti - mouse / rat - tgf- , or apc - conjugated anti - mouse / rat - nrp-1 for 30 minutes at 4c in the dark based on the same cell number . for determination of intranuclear foxp-3 , cd4cd25tregs were suspended in 1 ml fixation / permeabilization solution for 2 hours at 4c in the dark . after washing cells with permeabilization buffer twice , cd4cd25tregs were stained with fitc - conjugated anti - mouse / rat - foxp-3 for 30 minutes at 4c in the dark . after washing cd4cd25tregs with phosphate - buffered saline ( pbs ) twice , cells were analyzed with flow cytometer ( bd biosciences , mountain view , ca ) . 5 101 10 cd4cd25tregs or cd4cd25 t cells were washed with pbs twice ; cells were suspended in 200 l binding buffer , followed by 10 l fitc - conjugated annexin - v to stain for 30 minutes at 4c or 15 minutes at 25c in the dark . 300 l binding buffer and 5 l pi were added to stain for 5 minutes at 25c in the dark again , and they were subjected to flow cytometric analysis by flow cytometer . the supernatants were collected for measuring the levels of ifn- , il-4 , tgf- , and il-10 by elisa kits , strictly according to the manufacturer 's protocols . the standard concentration curves for ifn- , il-4 , tgf- , and il-10 were plotted from 0 to 1000 pg / ml . data was represented as mean standard deviation ( sd ) and analyzed by spss 17.0 software with a one - way anova . survival rate in septic mice was evaluated by kaplan - meier via the log - rank test . as shown in figures 1(a ) and 1(e ) , cd4cd25tregs were isolated from spleens at 24 hours after clp , and it was found that , compared with the control group , expressions of nrp-1 and foxp-3 of cd4cd25tregs were significantly promoted by sepsis ( p < 0.01 ) . compared with low - grade septic group , the expressions of nrp-1 and foxp-3 were further promoted in mid- and high - grade septic groups ( p < 0.01 ) , and the difference between mid- and high - grade septic groups was not statistically significant ( p > 0.05 ) . as shown in figure 1(b ) , the survival rate of low- , mid- , and high - grade septic groups was 86.67% , 66.67% , and 33.33% , respectively . the differences between them were statistically significant according to kaplan - meier analysis ( p < 0.01 ) . in figures 1(a ) , 1(b ) , and 1(e ) , we noticed that sepsis promoted the expression of nrp-1 along with the severity of sepsis and the upregulation of cytoactivity of cd4cd25tregs . we used midgrade septic model to further observe the time - dependent impact of sepsis on the expression of nrp-1 and foxp-3 . as shown in figures 1(c ) and 1(f ) , compared with the control group , the expressions of nrp-1 and foxp-3 were significantly promoted at every point after clp ( p < 0.01 ) . compared with 12-hour group , expressions of nrp-1 and foxp-3 were markedly enhanced at 24 and 48 hours ( p < 0.01 ) , but they were downregulated at 72 hours , and the difference between them was not statistically significant ( p > 0.05 ) . the survival rate of 12- , 24- , and 48-hour groups was 93.33% , 66.67% , and 26.67% ( figure 1(d ) ) , respectively , and the differences between them were statistically significant according to kaplan - meier analysis ( p < 0.01 ) , but the difference between 48- and 72-hour ( 20% ) groups was not statistically significant ( p > 0.05 ) . as shown in figures 2(a ) , 2(b ) , and 2(c ) , midgrade septic model was used to evaluate the impact of nrp-1 on the negative immunoregulation of tregs at 24 hours after clp . compared with the control group , sepsis significantly upregulated the expression of foxp-3/ctla-4/tgf- on cd4cd25tregs and nrp-1cd4cd25tregs ( p < 0.01 ) , but the expression of foxp-3/ctla-4/tgf- on nrp-1cd4cd25tregs was significantly downregulated ( p < 0.01 ) . compared with cd4cd25tregs , as shown in figure 3 , with the midgrade septic model at 24 hours , compared with the control group , the apoptotic rates of cd4cd25tregs and nrp-1cd4cd25tregs were markedly reduced ( p < 0.01 ) , but the apoptotic rate of nrp-1cd4cd25tregs was increased ( p < 0.05 ) . in contrast to cd4cd25tregs , as shown in figure 4 , with the midgrade septic model at 24 hours , cd4cd25tregs , nrp-1cd4cd25tregs , and nrp-1cd4cd25tregs were cocultured with cd4cd25 t cells for 24 hours in a ratio of 1 : 1 , respectively . the production of il-10 and tgf- was significantly increased from cd4cd25tregs and nrp-1cd4cd25tregs compared with that of the control group ( p < 0.01 ) , especially for nrp-1cd4cd25tregs as compared with that of cd4cd25tregs ( p < 0.01 ) . however , il-10 and tgf- levels were significantly descended in nrp-1cd4cd25tregs ( p < 0.01 ) . cd4cd25tregs , nrp-1cd4cd25tregs , and nrp-1cd4cd25tregs were isolated from the midgrade septic model at 24 hours and cocultured with conventional cd4cd25 t cells for 24 hours with a ratio of 1 : 1 , respectively . compared with the control group , the proliferation ( figure 5(a ) ) of cd4cd25 t cells was significantly suppressed , and the apoptosis ( figures 5(b ) and 5(c ) ) of cd4cd25 t cells was significantly increased when cocultured with cd4cd25tregs and nrp-1cd4cd25tregs ( p < 0.01 ) , but the proliferation of cd4cd25 t cells was obviously upregulated , and the apoptosis of cd4cd25 t cells was obviously downregulated , when cocultured with nrp-1cd4cd25tregs ( p < 0.01 ) . compared with cd4cd25tregs , the proliferation of cd4cd25 t cells was further significantly inhibited , and the apoptosis of cd4cd25 t cells was further significantly increased when cocultured with nrp-1cd4cd25tregs ( p < 0.05 or 0.01 ) . as shown in figure 6 , cd4cd25tregs , nrp-1cd4cd25tregs , and nrp-1cd4cd25tregs were , respectively , isolated from the midgrade septic model at 24 hours and cocultured with cd4cd25 t cells for 24 hours in a ratio of 1 : 1 . in comparison to the control group , secretion of ifn- of cd4cd25 t cells was decreased , but release of il-4 was increased when cocultured with cd4cd25tregs ( p < 0.05 ) . nrp-1cd4cd25tregs significantly suppressed the secretion of ifn- and il-4 from cd4cd25 t cells as compared with the control group and cd4cd25tregs ( p < 0.05 or 0.01 ) . the formation of ifn- and il-4 from cd4cd25 t cells was significantly upregulated when cocultured with nrp-1cd4cd25tregs ( p < 0.01 ) . as shown in figure 7 , in vitro , the demethylation level of foxp-3-tsdr in nrp-1cd4cd25tregs was significantly increased in the stimulated lps compared with control group for 24 hours ( p < 0.01 ) ; recombinant nrp-1 polyclonal antibody had an obvious ability to downregulate the demethylation level of foxp-3-tsdr , and in a dose - dependent manner , especially 10000 g / ml ( p < 0.05 or 0.01 ) . foxp-3 , which is still the main intracellular marker for identification of tregs , is a distinctive transcriptional factor of tregs , and it is also critical for their function , differentiation , and maintenance [ 11 , 13 , 26 ] . our previous study demonstrated that a significantly increased expression of foxp-3 in tregs was positively correlated with the mortality of burn - induced septic mice [ 1417 ] . recently , nrp-1 , which was highly expressed on natural tregs but lowly expressed on induced tregs and not expressed on cd4cd25 cells , is a good marker to distinguish natural and induced tregs [ 2024 ] . more interestingly , another study showed a population of nrp-1tregs in human lymph nodes together with the positive expression of foxp-3 that inhibited the proliferative activity of t cells . in the current study , we first reported that sepsis per se markedly promoted the expression of nrp-1 on cd4cd25tregs in a grade- and time - dependent manner , the expression of nrp-1 on tregs was obviously correlated with the expression of foxp-3 and the mortality of clp - induced septic mice , and nrp-1 had prevented ability to preserve the negative immunoregulation of tregs in sepsis . it has been known that immune dysfunction of cd4 t - lymphocytes was one of the primary cellular mechanisms in sepsis - induced immunosuppressive state . immediate observation of specimens from spleen , thymus , and lung in septic patients who died in intensive care units , or those from murine clp model , showed a profound , progressive , apoptosis - induced loss of adaptive immunocytes , thereby resulting in a decrease in ability of producing antibodies and clearing life - threatening pathogens [ 6 , 7 , 2931 ] . it is well known that the activated cd4 t cells can mainly differentiate into th1 and th2 , and they mainly produced ifn- and il-4 , respectively . a shift to th2 response was noted to be corroborated sepsis - induced immunosuppression , and the secretion of th1 associated cytokines was thereby impaired , and , on the other hand , the secretion of th2 associated cytokines was increased during sepsis , and these phenomena were obviously correlated with the outcome of septic complications [ 7 , 31 ] . t cells are anergic when they respond to their specific antigens , which are identified as failure to proliferate or secrete cytokines . in the present study , the results suggested that nrp-1cd4cd25tregs had the strongest ability to inhibit the proliferation and increase the apoptosis , as well as inhibiting the cytokine secretion of cd4cd25 t cells , but nrp-1cd4cd25tregs had opposed ability on cd4cd25 t cells ; thus , we conjectured that nrp-1cd4cd25tregs had a potential ability to prevent cellular immunosuppression in sepsis . in the development of sepsis , tregs can mainly inhibit the activation of t - lymphocytes , especially cd4 t - lymphocytes through various suppressive mechanisms , including inhibitory cytokines ( such as il-10 and tgf- ) and cell - to - cell contact ( such as ctla-4 and tgf- ) , inhibition of cytolysis ( especially granzymes ) , downregulation of metabolic disruption , and suppression of maturation and function of antigen presenting cells , as well as upregulation of antiapoptotic ability [ 12 , 1417 ] . accumulated evidence has shown that a combination of foxp-3 , ctla-4 , tgf- , and inhibitory cytokines ( il-10 and tgf- ) might serve as active markers for tregs in the process of sepsis [ 6 , 7 , 13 , 29 , 30 ] . we used a midgrade septic model reproduced 24 hours after clp to investigate the impact of nrp-1 on the negative immunoregulation of tregs in the setting of sepsis . it was found that sepsis could obviously upregulate the negative immunoregulation of cd4cd25tregs and nrp-1cd4cd25tregs , especially nrp-1cd4cd25tregs , but downregulated the negative immunoregulation of nrp-1cd4cd25tregs , which correlated with the expressions of foxp-3/ctla-4/tgf- and the apoptotic ability , as well as the secretion of il-10 and tgf-. we have reported that the percentage and stability of tregs were higher in septic patients and mice septic models than in those without sepsis . a reduction in the percentage and stability of tregs was accompanied by an improvement in survival rate and immune dysfunction of t - lymphocytes in septic mice [ 1417 ] . it has been documented that the stability of tregs includes the stability of foxp-3 expression and negative immunoregulatory function in sepsis , which is crucially dependent on the demethylation status of the foxp-3-tsdr . in the current study , we found that recombinant nrp-1 polyclonal antibody could downregulate the demethylation of foxp-3-tsdr in the presence of lps ; thus , nrp-1 could represent a new potential therapeutic target , at least via regulating the stability of tregs , for the study of immune regulation in sepsis . nrp-1cd4cd25tregs showed strong resilience to apoptosis and cytokine secretive ability in sepsis , and they possessed a strong ability to inhibit the proliferation and increase the apoptosis , as well as secreting cytokines of cd4cd25 t cells . recombinant nrp-1 polyclonal antibody had the ability to downregulate the demethylation of foxp-3-tsdr in the presence of lipopolysaccharide ( lps ) . nrp-1cd4cd25tregs might exhibit primary negative immunoregulation in sepsis , and nrp-1 could represent a new potential therapeutic target for the study of immune regulation in sepsis .

regulatory t cells ( tregs ) appear to be involved in sepsis - induced immune dysfunction ; neuropilin-1 ( nrp-1 ) was identified as a surface marker for cd4+cd25+tregs . in the current study , we investigated the negative immunoregulation of nrp-1highcd4+cd25+tregs and the potential therapeutic value of nrp-1 in sepsis . splenic cd4+cd25+tregs from cecal ligation and puncture ( clp ) mouse models were further segregated into nrp-1hightregs and nrp-1lowtregs ; they were cocultured with cd4+cd25 t cells . the expression of forkhead / winged helix transcription factor-3 ( foxp-3 ) , cytotoxic t - lymphocyte associated antigen-4 ( ctla-4 ) , membrane associated transforming growth factor- ( tgf-m+ ) , apoptotic rate , and secretive ability [ including tgf- and interleukin-10 ( il-10 ) ] for various types of tregs , as well as the immunosuppressive ability of tregs on cd4+cd25 t cells , were determined . meanwhile , the impact of recombinant nrp-1 polyclonal antibody on the demethylation of foxp-3-tsdr ( treg - specific demethylated region ) was measured in in vitro study . sepsis per se markedly promoted the expression of nrp-1 of cd4+cd25+tregs . foxp-3/ctla-4/tgf-m+ of nrp-1hightregs were upregulated by septic challenge . nrp-1hightregs showed strong resilience to apoptosis and secretive ability and the strongest immunosuppressive ability on cd4+cd25 t cells . in the presence of lipopolysaccharide ( lps ) , the recombinant nrp-1 polyclonal antibody reduced the demethylation of foxp-3-tsdr . nrp-1hightregs might reveal primary negative immunoregulation in sepsis ; nrp-1 could represent a new potential therapeutic target for the study of immune regulation in sepsis .

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

PMC3181937

in the past , psychiatric diseases have been treated pharmacologically with broad - profile medication - the socalled shotgun method . in the same way that a shotgun fires many pellets at once , psychiatric medication can impact on many different neurotransmitter systems . due to this profile , many of these drugs , such as tricyclic antidepressants ( tcas ) or first - generation antipsychotics ( fgas ) caused severe undesirable side effects , which were held responsible for poor compliance and discontinuation of the prescribed medication . during the last two decades , new drugs have surfaced with fewer shotgun side effects because of their particular pharmacodynamic design targeted against one single and very specific molecule . in this context , selective serotonin reuptake inhibitors ( ssris ) should be mentioned here as an example of an antidepressant agent acting solely on one neurotransmitter system and within the serotonergic system on one distinct transporter molecule . the same holds true for second - generation antipsychotics ( sgas ) displaying only few side effects due to less rigid inactivation of dopamine receptor type 2 ( dr2 ) and therefore fewer extrapyramidal motor symptoms recalling parkinsonism . nevertheless , we are still struggling with inefficacious medication , since only about one third of antidepressant agents work in a given patient , meaning that one has to try on average three different medications in order to alleviate this patient 's symptoms . for schizophrenia sometimes , the individual situation seems even worse than in the field of affective disorders . as mentioned above , this article emphasizes the link between disturbed adult neurogenesis ( an ) and affective disorder . the case seems to be much more evident here than in schizophrenia , although decreased neural stem cell proliferation in the dentate gyrus ( dg ) of the hippocampus has been demonstrated in postmortem human brain from schizophrenic patients . stem cells can be characterized by two fundamental qualities : first , they have the capacity for unlimited selfrenewal , and second , they can produce at least one type of highly differentiated descendants . this particular cell division is termed asymmetrical : in general , each stem cell division gives rise to one stem and one committed somatic daughter cell . stem cells are single cells that , once developed , self -renew for the lifetime of the organism . these stem cells should be distinguished from transient progenitor cells , which have a limited self - renewal lifespan . some steps earlier during the embryonic period , cells become gradually restricted to distinct pathways of differentiation . this process includes modification of their developmental potential ; they become pluripotent ( many , several ) . the major difference between totipotency and pluripotency is that an embryonic stem cell ( es cell ) which is by definition pluripotent , can only form cells which constitute the embryo itself but not the placenta . early es cells can be taken from the embryo and grown in vitro . when retransferred into the embryo es cells also play a central role in the generation of transgenic animals such as knockout mice . multipotent stem cells in the brain ultimately give rise to all different types of neuronal and non - neuronal cells in the central nervous system . because they are capable of generating the entire progeny of a given tissue , some investigators have termed these multipotent stem cells moreover , there is controversy as to whether neural stem cells can actually remain viable during the entire lifespan . adult forebrain neural stem cells were discovered in 1992 in the adult remnant of the embryonic brain germinal zone surrounding the lateral ventricle . evidence for their participation in repopulating the adult lateral ventricular subependyma following irradiation led to the hypothesis that neural stem cells also exist after the embryonic and fetal period , similarly to hematopoietic stem cells . hematopoietic stem cells in adult animals can restore different blood cell types . for a long time it has been thought that once a cell had been programmed to produce a particular tissue , its fate was sealed and it could not reprogram itself to form another tissue . conirarily to this view , reactivation of dormant genetic programs appears to work under certain circumstances . intriguingly , stem cells from brains of adult mice have been shown to possess the potential to become functional blood cells . similar results for the inverse case were reported , ie , multipotential mesenchymal cells transformed into neural cells - astrocytes in this case . according to fred h. gage , the term neural stem cell should therefore be used with caution to describe cells that a ) generate neural tissue , b ) are capable of selfrenewal , and c ) give rise to cells other than themselves through asymmetric cell division . in fact , when the issue of lineage specificity of adult neural stem and progenitor cells is considered , there is abundant reason for caution regarding the term neural stem cell . the ability of bone marrow stem cells to generate astrocytes , the finding that astrocyte - like cells in the subependyma are neural stem cells , and that oligodendrocyte - precursors contribute neural stem cell - like cells - all these findings soften the theoretical distinction between stem and progenitor cells . all this makes it really difficult to decide which type of stem cells one should use for transplantation . even during the process of grafting stem cells they can still start to differentiate and become more restricted progenitors due to cell - cell contacts or influence of adhesion inside the syringe . further , neural stem cell research suffers dearly from the lack of an antibody specifically identifying neural stem cells . putative stem cells need to differentiate into their derivative neural cell subpopulations before one can positively identify them as regular neural stem cells . in the past there are certain advantages of adult over embryonic stem cells in that the former may be easier to manage . for example , when injected subcutaneously into immunocompromised mice they grow into teratomas , tumors consisting of numerous cell types ranging from gut to skin . before applying these cells in humans , generation of the desired cell types should therefore be ensured without undesired side effects or unwanted cell populations , respectively . here , it seems that adult stem cells are better behaved , since they do not differentiate spontaneously . instead this can be induced by applying appropriate growth factors . however , adult stem cells have a different drawback , in that they seem to lose their ability to divide and differentiate after some time in culture . maybe in the end ethical considerations will also convince the scientific community to follow the adult stem cell rather than the es cell track . compared with embryonic or fetal stem cells , adult stem cells pose fewer ethical problems because they can be obtained from sources other than embryos or aborted fetuses . even postmortem human tissue can yield neural stem cells . in consensus with frank e. young the public , as well as governmental authorities , should enter the process of unbiased dialogue , in order to establish the principles according to which research needs to be conducted . as of now , we should consider the human species an appropriate source for scbi . having said this , we should take into account possible chimerae of animals with human cells in their brains , and above all with possible human behavior . another issue to be ruled out is to prevent striking behavioral traits after scbi . in parkinson 's disease patients it has been shown that after l - dopa treatment some patients responded with pathological gambling , since dopamine sustains the reward system . one could easily imagine a scenario like this in a patient after scbi . in this case , it might not be as easy to lower the dopamine production as it is with cessation of the medication . as mentioned above , grafting hematopoietic stem cells has already become a conventional clinical tool in the treatment of certain types of leukemia . currently it can only be hypothesized that transplantation of neural stem cells has potential for treating brain disease . although all these obstacles do exist , the main target for the research on neural stem cells must be to restore regular neural function in areas where cells have died or lost their physiologic behavior . clinicians are eager , for example , to transplant nscs into patients suffering from parkinson 's disease , multiple sclerosis , or spinal cord injuries , although it is not clear so far which is the appropriate cell to transplant - the cns neural stem cells , the actual neurons , or intermediate progenitors between the two . thus , in neurodegenerative diseases it is important to first determine the rules of transplantation of stem , progenitor , and mature cells , as well as to determine the sites into which the transplants must be located . in parkinson 's disease we do not know whether cells should be placed into the substantia nigra , or striatum , or both . so we continue wondering which particular region of the abovementioned brain areas should be chosen - eg , the ventral part or the dorsal putamen in only one hemisphere or both . the variability in surgical methods between centers which perform stem cell transplantation makes it very difficult to assess the results of this procedure . still , if we get to the point where all these issues are resolved , we will have to find better ways to secure the long - term survival of the grafted cells . so far , the de novo generated dopaminergic neurons have only a limited lifespan in animal studies . finally , the source of the cells should be clarified , since we need huge amounts of human fetal tissue for transplantation . although there has been much progress achieved over the last few years , especially in long - term proliferation and dopaminergic differentiation of progenitor cells , we still have to invest much effort in finding alternative methods , such as in vivo mobilization of dormant neural stem cells , which seems to be a more useful concept . in the light of recent findings such as loss of total cell volume in certain brain areas observed in depressed patients ( see below ) , we can heretically define depression as a neurodegenerative disease . as we will discuss , this particular illness does not necessarily require stem cell transplantation . instead , it may be possible to replenish missing neurons by regulating the microenvironment surrounding the putative sites of neural stem cells where neurogenesis takes place . lessons from recent findings in pathophysiology might open up a broad research avenue with the ultimate goal of treating depression . in a who survey , depression and manic - depressive illness will still rank among the top 10 causes for death in the year 2010 . certainly not all forms of depression eventually lead to death by suicide , but even milder courses of this illness may lead to severe incapacitation of millions of people worldwide . in particular , it impairs reintegration into their familiar social environment and into the working process . accumulating evidence from neurobiology suggests that distinct biochemical processes are derailed in a large number of depressed subjects . cellular and molecular adjustments following stress seem to play - key roles in onset and propagation of mood disorders . to most of us , stress is a beneficial response of our neuronal systems to acute challenges of the exterior world . to put it simply , it is more beneficial for a rabbit to become stressed and flee when it sees a fox approaching . however , severe or repeated stress can lead to detrimental effects on regular neural function . neural plasticity is a term which involves interneuronal communication and adaptation in order to give the appropriate responses to stress or aversive stimuli . one example of such a response could be changes in neurogenesis - the generation of new neurons . dysfunction of adequate regulatory processing due to severe or chronic stress is capable of disturbing neural plasticity . medical treatment with antidepressant agents may bring back regular function of neural systems by influencing neural plasticity : antidepressants require long - term administration , while blockade of the reuptake of serotonin ( 5-ht ) and/or norepinephrine as their most common and initial mode of action is fairly rapid . there is a process whereby neurons can adapt to and regain plasticity while the local biochemical environment is changing due to the application of antidepressants . thus , the long - term mode of action of antidepressant medication seems much more dynamic and complex than just up- or downregulation of synaptic levels of monoamines . g golgi stained hippocampal neurons with his novel silver impregnation technique , which became known as the golgi procedure . since then a great number of neuropsychiatrie phenomena have been studied in the hippocampal formation . the relatively- simple organization - pyramidal neurons in the hippocampus proper and the granule cells of the dentate gyrus are arranged in single , densely packed cell layers - is one of the major reasons why the hippocampus has frequently been used as a cytoarchitectural model of the cortex . recent findings in volumetric neuroimaging studies make a strong case that biochemical changes in the brain carry morphological sequels . so far we have learned that gray matter volumes are diminished in depressed patients and in post - traumatic stress disorder patients in the medial and orbital prefrontal cortex , the mesiotemporal cortex , and the ventral striatum , and are accompanied by an enlargement of the third and the lateral ventricles . significant reduction in numbers of nonpyramidal neurons in the ca2 area of hippocampus was reported in postmortem studies of bipolar disorder . also in regions other than hippocampus , there may be a decline in brain region volume and total cell number . elevation of cortisol levels in the elderly correlates with reduced hippocampal volume , and is associated with memory deficits . patients with depression have a functional deficit of the hypothalamic - pituitary - adrenal ( fifa ) axis . hippocampal neurons are reported to be damaged by exposure to stress or activation of the iipa axis and elevation of glucocorticoids . taken together , this overview of morphologic evidence strongly supports a functional link between changes at the molecular levels and morphology . the task of future research could be to develop strategies allowing the diseased hippocampus or other affected brain structures to regain regular morphology and function . focusing on neurogenesis - which is defined as a series of events including proliferation of a neural precursor or stem cell that results in appearance of a new neuron - may be a systematic as well as pragmatic way to proceed . localization of pluripotent progenitor cells and thus neurogenesis appears to be restricted to certain brain regions , in particular , the subventricular zone ( svz ) and the subgranular layer of the dentate gyrus of the hippocampus . neurogenesis in the adult mammalian brain is regulated by genetic and environmental factors - all leading to the exciting possibility of pharmacological regulation of neurogenesis in the adult brain , and eventually of the disease - related pathophysiological changes . one of the mainstay therapies in the treatment of recurrent mood disorders , lithium , ranks among such pharmacologic candidates . we now know that besides its role in cell cycle control , bcl-2 functions as a neurotrophic factor , since bcl-2 promotes axon regeneration as well as neurite and axonal outgrowth . in general , neurotrophic factor signaling is mediated both by the phosphatidyl - inositol-3-kinase pathway and activation of the map ( mitogen - activated kinase ) cascade . this is very likely to involve the camp responsive element binding protein ( creb ) . creb is attractive to many researchers because it appears in some way required for long - term memory . creb may increase the integrity and functional plasticity of granule cell neurons assuming that creb is a critical determinant of neural plasticity as well as cell survival . one putative gene target of creb - and thus of chronic antidepressant treatment - is brain - derived neurotrophic factor ( bdnf ) . there is a functional camp responsive element in the exon iii promoter of the bdnf gene . in the light of this , it is not surprising that local infusion of bdnf in the hippocampus produces an antidepressant effect . in vitro , activation of the camp system upregulates bdnf expression in hippocampal cells . additionally , bdnf expression effects neuronal depolarization and activation of voltage -dependent calcium channels . these alterations at the synaptic level underlie the influence of bdnf on long - term potentiation . this underscores the central role of bdnf in neurogenesis considering the pivotal role attributed to bdnf in lineage differentiation of neural stem cells . another key player in the pathophysiology and treatment of depression , the biogenic amine 5-ht , should not be neglected , since 5-ht is one of the most extensively studied neurotransmitters of the central nervous system . moreover , novel findings indicate that 5 - 1 it is particularly relevant to neurogenesis in the hippocampus ( figure 1 ) , because in adult rats it has been shown that decreased 5-ht lowers the rate of neurogenesis in the dentate gyrus of hippocampus . historically , 5-ht was first described as a serum component augmenting smooth muscle contraction . in such non - neural systems all serotonergic fibers originate in the brain stem raphe nuclei . by way of extensive synaptic connections of the serotonergic fibers , 5-ht contributes to many physiologic functions such as endocrine and circadian rhythms , food intake , sleep , reproductive activity , and motor function , as well as cognition , mood , and anxiety . in the brain we currently know of 16 different cloned receptor types and subtypes , but it can be expected that their number will grow even further in the near future . in contrast to the multitude of 5 - 1 it receptors , there is only a single 5-ht transporter ( 5-htt ) responsible for the reuptake of 5-ht into serotonergic neurons after its release into the synaptic cleft . as our own studies have shown , 5 -i itt does not have a large impact on neurogenesis . a possible role for 5-ht as direct mediator of granule cell generation is currently discussed , since elevated 5-ht levels in the hippocampus increase the rate of proliferation of granule cell precursors . epidermal growth factor ( egf ) is believed to exert an essential function on the generation and maintenance of neural stem cells . it is therefore not surprising that in non - neural systems , egf and 5-ht can augment the rate of cell proliferation in a synergistic manner . thus , chronic administration of 5-iit - selective reuptake inhibitors , clinically used as antidepressants , leads to upregulation of bdnf nirna . as already mentioned above , 5-ht exerts its action through a large family of receptors in the periphery and throughout the cns . a possible role for the 5-iit1a receptor in the modulation of anxiety and depression , as well as in the mode of action of anxiolytic and antidepressant drugs , has been suspected for many years . research regarding 5-ht1a receptor has shown that the effect of antidepressants upregulating extracellular serotonin levels worked via the 5-ht1a receptor subtype , thus opening a link between our in vitro system , neurogenesis , and clinical relevance in terms of affective disorders . althogh the serotonin hypothesis of depression is very attractive in this regard , it should not be omitted here that there are additional compelling findings dealing with other neurotransmitting systems , eg , supporting cholinergic mechanisms . riederer et al came to the conclusion that a brain area - specific imbalance of neurotransmitters leads to the clinically different manifestations of depression . the loss of regional interneuronal homeostasis must not necessarily affect huge brain areas ; it might be only limited to certain small and circumscripted regions in the brain . as a consequence , clinicians should be able to choose the pharmacologically appropriate medication for the affected brain region . stem - cell maintenance and generation take place in a distinct microenvironment where appropriate external signals can best exert their regulatory function on these cells . since components of regular stem - cell maintenance like bdnf are also implicated in mechanistic models characteristic of mood disorders , they thus offer new targets for pharmacologic intervention in neuropsychiatrie disease . more thorough knowledge about this complex connection mayhelp us render antidepressant treatment more efficient and reduce the undesirable side effects that impair patient compliance . so far there is no stem - cell - based approach really on the horizon for treating depression or any other psychosis .

exploring stem ceils is a fascinating task , especially in a discipline where the use of stem cells seems far - fetched at first glance , as is the case in psychiatry . in this article we would like to provide a brief overview of the current situation in relation to the treatment of mental diseases . for reasons that we will explain , this review will focus on affective disorders . the following section will give a more detailed account of stem - cell biology , including current basic science approaches presenting in - vivo andin - vitro techniques . the final part will then look into future perspectives of using these stem cells to cure mental illnesses , and discuss the related challenges and opportunities .

Past and current status Some stem cell basics Pathophysiologic models of depression The role of the hippocampus Concluding remarks

PMC3968737

in the past few years , prevalence of obesity in urban youth has reached striking levels in india as a result of which children are exposed to health risks . a sedentary lifestyle is one of the major contributors to obesity that increases the risk of cardiovascular disease ( cvd ) in children in the latter further , high adiposity and sedentary lifestyle in childhood is associated with early development of dyslipidemia , insulin resistance and arterial stiffness predicting the future risk of cardiovascular ( cv ) disease in adulthood . evidence suggests that the atherosclerotic process starts in childhood and vascular status is abnormal even in overweight children and adolescents . studies have reported inverse and no correlation between physical activity and arterial stiffness in children . thus , limited work on the relationship between physical activity and arterial health in children has provided contradictory findings . hence , a better understanding of structural and functional changes of arteries and its association with activity and adiposity in children will help in instituting preventive measures for atherosclerotic risk . waist circumference ( wc ) , a measure of abdominal obesity , is more closely related to cardiovascular ( cv ) risk factors than bmi in children and adults . wc is also considered a key component in defining the metabolic syndrome . the increasing prevalence of obesity and metabolic syndrome worldwide in recent years demonstrates urgent need for appropriate cutoff points for wc in children and adolescents for early intervention . ethnicity - specific cutoffs of wc for western adults and indian 's have been suggested . similar cutoffs have been provided by nhanes - iii for us children . however , for early detection of cardio - metabolic risk , such cutoffs in asian children are not available . further , the fact that body fat percentage and waist circumference ( central adiposity ) in indian children are high , makes them more vulnerable for arterial abnormalities ; thus , the specific objectives of the present study were to explore the association of adiposity and physical activity with arterial stiffness in indian obese children and to propose optimal wc cutoff points for indian children which correspond to the 90 wc percentiles of nhanes - iii for children in order to predict risk of arterial stiffness . in a cross - sectional study , a total of 250 children and adolescents in the age range of 6 to 17 years ( mean age : 11.4 2.8 year ) were selected randomly from private schools and routine health checks from tertiary care hospitals in pune city and around suburbs , india on voluntary basis during 2008 - 2009 thus , representative sample . from the list of schools in pune city , two schools were randomly selected then further using cluster sampling method class was selected and in a class all the children were enrolled for the study . for selection of children from health checks randomization was done by computer generated random number list . using the standard deviations of previous reported arterial and metabolic measurements ( triglycerides , insulin , bmi , and carotid intima media thickness ( cimt ) ) in indian youth ; sample size of 250 was estimated cross - sectionally to have overall power of the study to be 0.81 with level of significance 0.05 . children with frank type i or ii diabetes mellitus ( dm ) and any endocrine disorders other than nutritional obesity ( high body mass index but no endocrine issues ) were excluded from the study . children and adolescents who were suffering or those who had suffered with endocrine diseases [ other than impaired glucose tolerance ( igt ) ] , craniopharyngioma or those undergoing medication that alters blood pressure or glucose or lipid metabolism were excluded . children with frank type ii diabetes were also excluded . to rule out endocrinological causes of obesity , the research protocol was approved by the ethics committee of hirabai cowasji jehangir medical research institute ( hcjmri ) , pune , india . clinical examination of all children was performed by a pediatrician to assess their health status . a written informed consent was obtained from the parents and assent was obtained from children prior to actual commencement of the study . body mass index [ bmi ] was computed according to the current available indian growth monitoring guidelines , the reference values at 85 and 95 percentile of nationally representative data were used to define overweight and obesity respectively . activity was assessed by using the activity questionnaire , which was adapted for indian children and adolescents lifestyle . each participant was asked to report the usual amount of time spent in different daily activities . time spent by the subjects in personal activities , school activities , reading and commuting was considered as light activity . time spent in sports activities ( e.g. , walking , jogging , swimming , dancing , and playing outdoor games ) was considered as moderate activity . the questionnaire was validated against a 24 hour activity recall on a pilot sample of 15 children . test - retest reliability of these measures was calculated ( intra - class correlation coefficient , r = 0.94 , p < 0.01 for inactivity ; r = 0.92 , p < 0.01 for light activity ; r = 0.99 , p < 0.01 for moderate activity ) dual energy x - ray absorptiometry ( dexa ) was used for measurement of body fat percentage . dexa measurements were performed using lunar dpx - pro total body pencil beam densitometer ( ge healthcare , wisconsin , usa ) using a medium mode scan ( software encore 2005 version 9.30.044 ) for total body fat percentage . measurements were standardized in children and adolescents by running daily quality assurance scans and reproducibility was established . the amount of effective dose of radiation exposure for a whole body dexa is 0.02 sv which is safe ( personal communication with dr qi zhou , ge lunar , evidence based marketing manager asia lunar , january 2010 ) . blood pressure was measured in the right arm with child lying down quietly for 10 min . measurements were made by auscultation with a mercury - column sphygmomanometer and a cuff appropriately sized for the arm size of the participant . arterial measurements were carried out at right carotid artery using an aloka alfa-10 ultrasound apparatus ( echotracking ) ( model ssd- 10 ; no : m00542 ; 2007 ; aloka co. ltd . japan ) to track the vessel wall motion and automatically construct the diameter change curve in real time by a radiologist . the 4 physiological parameters ; stiffness ( ) , elastic modulus ( ep ) , arterial compliance ( ac ) and pulse wave velocity ( pwv ) of the right common carotid artery along with cimt were analyzed in the present study . a chinese study has provided reference stiffness data for each gender across age groups ( < 10 years , 10 - 19 and so on ) considering the comparable ethnicity and no availability of indian cutoffs , stiffness parameters in the present study were compared with age - gender specific healthy reference chinese for determining high and low pwv . analyses were performed using spss software for windows ( version 11.0 , 2001 , spss inc , chicago , il ) . descriptive statistics for general characteristics and arterial parameters were calculated for 2 groups of normal weight and overweight / obese children . differences in means amongst normal weight and overweight / obese children were tested using independent student t - test . pearson 's correlation coefficients were computed to explore association between anthropometric , stiffness and physical activity parameters . as pwv is considered as an important marker for evaluation of early functional changes of the carotid artery in children and adolescents , multivariate regression analysis carried out with pwv as dependent variables and bmi , waist circumference , body fat and moderate physical activity as independent variable . waist z scores were computed by standardizing the variable ( waist circumference ) and by regressing it onto age to account for any age related differences . regressions were carried out separately for both the genders . the standardized residual ( z - score ) for waist circumference were then used as a test variable in receiver operating characteristic curve ( roc ) curve analyses . roc curve is a plot of the true positive rate ( sensitivity ) against the false positive rate ( 1-specificity ) across range of values from the diagnostic test . the decision threshold is the criterion value with the highest accuracy that maximizes the sum of the sensitivity and specificity . roc analysis was utilized to determine cutoff values of waist circumference that minimize the total number of misclassification errors and to provide an evaluation of the global performance of the waist circumference cutoffs to discriminate between those with or without risk of high pwv ( stiffness ) . body mass index [ bmi ] was computed according to the current available indian growth monitoring guidelines , the reference values at 85 and 95 percentile of nationally representative data were used to define overweight and obesity respectively . activity was assessed by using the activity questionnaire , which was adapted for indian children and adolescents lifestyle . each participant was asked to report the usual amount of time spent in different daily activities . time spent by the subjects in personal activities , school activities , reading and commuting was considered as light activity . time spent in sports activities ( e.g. , walking , jogging , swimming , dancing , and playing outdoor games ) was considered as moderate activity . the questionnaire was validated against a 24 hour activity recall on a pilot sample of 15 children . test - retest reliability of these measures was calculated ( intra - class correlation coefficient , r = 0.94 , p < 0.01 for inactivity ; r = 0.92 , p < 0.01 for light activity ; r = 0.99 , p < 0.01 for moderate activity ) dual energy x - ray absorptiometry ( dexa ) was used for measurement of body fat percentage . dexa measurements were performed using lunar dpx - pro total body pencil beam densitometer ( ge healthcare , wisconsin , usa ) using a medium mode scan ( software encore 2005 version 9.30.044 ) for total body fat percentage . measurements were standardized in children and adolescents by running daily quality assurance scans and reproducibility was established . the amount of effective dose of radiation exposure for a whole body dexa is 0.02 sv which is safe ( personal communication with dr qi zhou , ge lunar , evidence based marketing manager asia lunar , january 2010 ) . blood pressure was measured in the right arm with child lying down quietly for 10 min . measurements were made by auscultation with a mercury - column sphygmomanometer and a cuff appropriately sized for the arm size of the participant . arterial measurements were carried out at right carotid artery using an aloka alfa-10 ultrasound apparatus ( echotracking ) ( model ssd- 10 ; no : m00542 ; 2007 ; aloka co. ltd . japan ) to track the vessel wall motion and automatically construct the diameter change curve in real time by a radiologist . the 4 physiological parameters ; stiffness ( ) , elastic modulus ( ep ) , arterial compliance ( ac ) and pulse wave velocity ( pwv ) of the right common carotid artery along with cimt were analyzed in the present study . a chinese study has provided reference stiffness data for each gender across age groups ( < 10 years , 10 - 19 and so on ) considering the comparable ethnicity and no availability of indian cutoffs , stiffness parameters in the present study were compared with age - gender specific healthy reference chinese for determining high and low pwv . analyses were performed using spss software for windows ( version 11.0 , 2001 , spss inc , chicago , il ) . descriptive statistics for general characteristics and arterial parameters were calculated for 2 groups of normal weight and overweight / obese children . differences in means amongst normal weight and overweight / obese children were tested using independent student t - test . pearson 's correlation coefficients were computed to explore association between anthropometric , stiffness and physical activity parameters . as pwv is considered as an important marker for evaluation of early functional changes of the carotid artery in children and adolescents , multivariate regression analysis carried out with pwv as dependent variables and bmi , waist circumference , body fat and moderate physical activity as independent variable . waist z scores were computed by standardizing the variable ( waist circumference ) and by regressing it onto age to account for any age related differences . the standardized residual ( z - score ) for waist circumference were then used as a test variable in receiver operating characteristic curve ( roc ) curve analyses . roc curve is a plot of the true positive rate ( sensitivity ) against the false positive rate ( 1-specificity ) across range of values from the diagnostic test . the decision threshold is the criterion value with the highest accuracy that maximizes the sum of the sensitivity and specificity . roc analysis was utilized to determine cutoff values of waist circumference that minimize the total number of misclassification errors and to provide an evaluation of the global performance of the waist circumference cutoffs to discriminate between those with or without risk of high pwv ( stiffness ) . children and adolescents in the present study were classified as normal weight , overweight and obese according to the 85 and 95 reference bmi percentiles of indian children . table 1 presents anthropometric characteristics and activity pattern of the study population across 2 groups of normal weight and overweight / obese children . mean age of normal weight and overweight / obese children was similar ( p > 0.1 ) . amongst anthropometric parameters , average weight , body mass index , waist and hip circumference , waist to hip ratio and waist to height ratio were significantly higher in overweight / obese children than normal weight children ( p < 0.05 ) [ table 1 ] . mean total body fat percentage was significantly higher in overweight / obese children in comparison to normal weight children ( p < 0.05 ) . as there is no indian reference database for body fat percentage , western cutoffs for body fat have been used . high adiposity was defined as body fat percentage greater than the 95 percentile of mccarthy 's body fat reference database for each yearly age - sex group . moreover , children when classified using mccarthy body fat cutoff , 37.1% normal weight children and 98.2% overweight / obese children had high adiposity . median time spent in sleep 480 min ( 8 h ) , personal work ( 30 min ) and average class room hours in school and reading hours was similar in both normal weight and overweight / obese children ( p > 0.1 ) . time spent in tv viewing was considered as inactivity , median time spent in television viewing was around 2 h 95ci ( 107 - 130 ) in overweight / obese which is suggestive of their sedentary behavior as compared to 1 h 95ci ( 77 - 102 ) in normal weight children . median moderate activity ( walking / jogging and sports and exercise ) was significantly higher in normal weight than overweight / obese children ( p < 0.05 ) [ table 1 ] . moreover , when the time spent in exercise was classified further as per cdc guidelines ; it was observed that only 36% overweight / obese children reported exercise for at least 60 min daily whereas ; 61% normal weight children reported exercise for at least 60 min [ table 1 ] . characteristics of the study population table 2 gives arterial parameters of the study population for the normal weight and overweight / obese children . significantly ; higher systolic and diastolic blood pressure was observed in overweight / obese children than normal weight children ( p < 0.05 ) . mean arterial parameters viz ; carotid intima media thickness , stiffness index , elasticity modulus and pulse wave velocity were significantly higher while arterial compliance was significantly lower in overweight / obese children than normal weight children ( p < 0.05 ) indicating cv risk in overweight children . arterial parameters of the study population correlation analyses revealed positive association of body fat percentage with pwv ( r = 0.46 ) , stiffness index ( ) ( r = 0.21 ) and ep ( r = 0.43 ) ( p < 0.05 ) whereas , ac showed a negative association with body fat percentage ( r = 0.25 ) envisaging higher risk of atherosclerosis in children with high body fat percentage . moderate physical activity was inversely associated with pwv ( r = 0.2 ) , ( r = 0.13 ) , ep ( r = 0.12 ) and positively with ac ( r = 0.12 ) which are markers of arterial stiffness ( p < 0.05 ) . similar correlations for light activity or inactivity could not be achieved . significant positive correlation was obtained for pwv and ep with other adiposity measures viz , bmi and waist circumference ( r = 0.5 ) ( p < 0.05 ) and negative correlation of ac with bmi and waist circumference ( r = 0.25 ) ( p < 0.05 ) demonstrating the importance of central adiposity and bmi in evaluating the risk of increased stiffness . waist to height ratio also showed positive association with pwv and ep ( r = 0.4 ) ( p < 0.05 ) and negative with ac ( r = 0.20 ) ( p < 0.05 ) however , similar associations with waist to hip ratio could not be achieved . additionally , change in pwv was examined across tertiles of moderate physical activity and body fat percentage [ figure 1 ] . pwv significantly increased with increasing body fat percentage for each tertile of moderate physical activity . also pwv decreased significantly with increasing moderate physical activity in each tertile of body fat percentage , thus illustrating increased risk of stiffness ( pwv ) with lack of moderate physical activity . to avoid the effect of co - linearity between waist circumference , bmi and body fat , separate multivariate regression analysis was carried out with pwv as dependent variable with physical activity and any one of the 3 variables , viz . ; analysis revealed , physical activity along with all the 3 measures of adiposity , i.e. waist circumference , bmi and body fat were significantly associated with stiffness ( pwv ) after adjusting for age ( adjusted r^2 = 0.21 , 0.24 , 0.22 respectively ) ( p < 0.05 ) . pwv by tertiles of body fat and moderate physical activity the cutoff value of waist circumference z - score yielding optimal sensitivity and specificity for predicting the risk of high pwv was found to be 0.43 for boys and -0.44 for girls . area under the curve ( auc ) was 0.60 ( 95% ci : ( 0.44 , 0.69 ) ) for boys and 0.78 ( 95% ci : 0.67 , 0.87 ) for girls . sensitivity was 78% and 87% and specificity was 51% and 70% for boys and girls respectively , [ figures 2 and 3 ] indicating correct classification of children above and below the waist circumference cutoff with respect to their presence or absence of high pwv . sensitivity 87% , specificity 70% , area under the curve 0.78 ( 95% ci : 0.67 , 0.87 ) roc curve of waist z score for boys with pwv . sensitivity 78% , specificity 51% , area under the curve is 0.60 ( 95% ci : 0.44 , 0.69 ) to facilitate application of the waist z score cutoffs , residual standard deviation of gender - specific regression of waist circumference on age was used . roc cutoffs of waist z scores for boys and girls were then converted into age specific waist circumference cutoffs using the 90 percentile of waist circumference from nhanes - iii [ table 3 ] . comparison of interpolated cutoffs of waist circumference with the existing 90 percentiles according to age and sex : nhanes - iii correlation analyses revealed positive association of body fat percentage with pwv ( r = 0.46 ) , stiffness index ( ) ( r = 0.21 ) and ep ( r = 0.43 ) ( p < 0.05 ) whereas , ac showed a negative association with body fat percentage ( r = 0.25 ) envisaging higher risk of atherosclerosis in children with high body fat percentage . moderate physical activity was inversely associated with pwv ( r = 0.2 ) , ( r = 0.13 ) , ep ( r = 0.12 ) and positively with ac ( r = 0.12 ) which are markers of arterial stiffness ( p < 0.05 ) significant positive correlation was obtained for pwv and ep with other adiposity measures viz , bmi and waist circumference ( r = 0.5 ) ( p < 0.05 ) and negative correlation of ac with bmi and waist circumference ( r = 0.25 ) ( p < 0.05 ) demonstrating the importance of central adiposity and bmi in evaluating the risk of increased stiffness . waist to height ratio also showed positive association with pwv and ep ( r = 0.4 ) ( p < 0.05 ) and negative with ac ( r = 0.20 ) ( p < 0.05 ) however , similar associations with waist to hip ratio could not be achieved . additionally , change in pwv was examined across tertiles of moderate physical activity and body fat percentage [ figure 1 ] . pwv significantly increased with increasing body fat percentage for each tertile of moderate physical activity . also pwv decreased significantly with increasing moderate physical activity in each tertile of body fat percentage , thus illustrating increased risk of stiffness ( pwv ) with lack of moderate physical activity . to avoid the effect of co - linearity between waist circumference , bmi and body fat , separate multivariate regression analysis was carried out with pwv as dependent variable with physical activity and any one of the 3 variables , viz . ; analysis revealed , physical activity along with all the 3 measures of adiposity , i.e. waist circumference , bmi and body fat were significantly associated with stiffness ( pwv ) after adjusting for age ( adjusted r^2 = 0.21 , 0.24 , 0.22 respectively ) ( p < 0.05 ) . pwv by tertiles of body fat and moderate physical activity the cutoff value of waist circumference z - score yielding optimal sensitivity and specificity for predicting the risk of high pwv was found to be 0.43 for boys and -0.44 for girls . area under the curve ( auc ) was 0.60 ( 95% ci : ( 0.44 , 0.69 ) ) for boys and 0.78 ( 95% ci : 0.67 , 0.87 ) for girls . sensitivity was 78% and 87% and specificity was 51% and 70% for boys and girls respectively , [ figures 2 and 3 ] indicating correct classification of children above and below the waist circumference cutoff with respect to their presence or absence of high pwv . sensitivity 87% , specificity 70% , area under the curve 0.78 ( 95% ci : 0.67 , 0.87 ) roc curve of waist z score for boys with pwv . sensitivity 78% , specificity 51% , area under the curve is 0.60 ( 95% ci : 0.44 , 0.69 ) to facilitate application of the waist z score cutoffs , residual standard deviation of gender - specific regression of waist circumference on age was used . roc cutoffs of waist z scores for boys and girls were then converted into age specific waist circumference cutoffs using the 90 percentile of waist circumference from nhanes - iii [ table 3 ] . comparison of interpolated cutoffs of waist circumference with the existing 90 percentiles according to age and sex : nhanes - iii present study , results imply that high adiposity ( in terms of higher body fat and higher waist circumference ) and low physical activity levels are associated with increased arterial stiffening in indian children and adolescents . pwv increases with increasing body fat percentage for each tertile of moderate physical activity . waist circumference was found to be a sensitive predictor of increased stiffness in children . in a similar study done on 10-year old children it was found that radial - femoral pwv was related to fat energy percentage and physical activity but not associated with body fat . in another study , on 970 healthy children , pwv measured between the brachium and ankle using a plethysmographic method was associated with increased age , bp , and heart rate but not bmi . these discrepancies in the results between studies may be attributable to differences in the methods used for the assessment of pwv . our findings suggest an association between decreased physical activity and higher body fat percentage and higher arterial stiffness which is on par with a cross - sectional study with respect to increasing pwv where , sakuragi et al . , have demonstrated an influence of cardio - respiratory fitness on pwv after adjusting for body fat in healthy pre - pubescent children . the influence of obesity and physical activity on arterial stiffness has also been reported in adults . kupari et al . , have also found a significant correlation between arterial distensibility and physical activity ( r = 0.45 ) . physical activity levels were evaluated and examined for association with metabolic syndrome by pan and pratt . pan and pratt have shown that the tertile with the lowest levels of physical activity had the highest prevalence of metabolic syndrome and the tertile with the highest levels of physical activity showed the lowest prevalence of metabolic syndrome , but the differences were not significant . another cross - sectional study in 9 - 15 yr old school children in denmark revealed decreasing odds ratio for clustered cardiovascular risk with ascending quintiles of physical activity . thus , the present study results revealing increasing stiffness with increasing tertile of body fat and decreasing tertile of physical activity are in line with the above finings . although , the underlying mechanisms by which increased adiposity promotes arterial stiffening can not be determined using cross - sectional data , several possibilities exists like increased bmi and adiposity are accompanied by increases in heart rate blood pressure , and intermediary cardiovascular metabolic risk factors such as insulin resistance and dyslipidemia . physical inactivity may be associated with both an increase in adiposity and increased arterial stiffness . arterial stiffness is determined by the properties of the arterial wall matrix and by vascular smooth muscle tone , and may be changed by an alteration in vascular smooth muscle tone caused by exercise . waist circumference is a better surrogate for body fat assessment and also one of the components in metabolic syndrome ; it has been identified as a risk factor for cvd in many studies . therefore , the efficacy of waist circumference in predicting the risk of arterial stiffness was identified using roc analysis . the standardized residual ( z - score ) of waist circumference was used as a test variable in roc curve analyses in order to account for the effect of age and gender . higher sensitivity obtained for waist circumference using roc analyses envisages waist circumference as putative marker of the risk of increased arterial stiffness ( pwv ) . further , it was observed that the proposed cutoff values for waist circumference were lower at all ages and for both the genders in comparison with those proposed by the nhanes - iii . these lower cutoffs are in line with lower cut - offs for bmi to define overweight and obesity for asian adults and children ( 23 and 28 vs 25 and 30 ) . also , in asian the risk association with diabetes and cardiovascular diseases occurs at lower levels of bmi when compared with the white population . this is attributed to body fat distribution ; asian indians tend to have more visceral adipose tissue , causing higher insulin resistance , despite having lean bmi . waist to height ratio was found to be significantly associated with pulse wave velocity and elasticity elucidating the importance of waist to height ratio in predicting increasing risk of arterial stiffness . average waist to height ratio in the present study is on par with swiss children ( 0.47 0.04 ) and also in agreement with the cutoff of 0.5 proposed by one indian study . a cross - sectional study in chinese children also has explored the optimal waist circumference value for predicting the cardiovascular risk . the findings suggest sensitivity and specificity ranging from 67% to 83% which is on par with the present study . however , further studies on larger data sets need to be performed to elucidate the most appropriate cutoffs for indian children and adolescents . also , with the lowered cutoffs proposed in the present study , validation is required . based on these findings , it can be concluded that , children with high adiposity and low physical activity are at higher risk of developing premature cardiovascular problems . the development of waist circumference percentiles and cut - offs in indian children is necessary because of ethnic differences in body composition .

objective : to explore association of adiposity and physical activity with arterial stiffness and to propose optimal waist circumference cutoffs , corresponding to 90th percentile of nhanes ( national health and nutrition examination survey ) for indian children and adolescents.materials and methods : data on weight , height , waist circumference , physical activity and right carotid artery intima - media - thickness ( cimt ) , pulse wave velocity ( pwv ) , elasticity modulus ( ep ) , stiffness index( ) , arterial compliance ( ac ) were assessed in 250 children ( 72 normal - weight and 178 overweight / obese ) aged 6 - 17 years from pune city , india . body composition was measured using dual energy x - ray absorptiometry.results:total , 37.1% normal - weight and 98.2% overweight / obese children had high adiposity ( > 95th body fat percentile ) . positive association of pwv and ep ( r = 0.5 ) also (r = 0.25 ) with bmi ( body mass index ) , waist circumference and body fat ( p < 0.05 ) was observed . physical activity was inversely associated with pwv ( r = -0.2 ) , (r = -0.13 ) , ep ( r = -0.12 ) and positively with ac ( r = 0.12 ) ( p < 0.05 ) . pwv significantly increased with increasing body fat for each tertile of physical activity ( p < 0.05 ) . regression analysis revealed waist circumference , bmi , body fat and physical activity as independent associates for pwv after adjusting for age ( p < 0.05 ) . the cutoff of waist circumference yielding sensitivity and specificity for predicting the risk of high pwv was ( 0.43 , 0.44 ) for boys and girls with sensitivity in boys ( girls ) of 78% ( 87% ) and specificity in boys ( girls ) 51% ( 70% ) . the observed cutoffs are less than the nhanes - iii cutoff values of waist circumference for 90th percentiles according to age and sex.conclusion:high adiposity and low physical activity are adversely related to arterial stiffness in indian children .

I M Anthropometric measures Physical activity Body composition Blood pressure Carotid arterial measurement Statistical analyses R Association of arterial parameters with adiposity and physical activity D

PMC4985254

the global epidemic in obesity and related disorders such as type 2 diabetes has fueled an explosion of interest in adipose ( fat ) cells . white fat is specialized to store energy in the form of triglycerides , an especially efficient method because this class of molecules is highly energetic and stored anhydrously . on fasting , the release of fatty acids and glycerol to provide fuel for the rest of the body occurs via enzymatic hydrolysis called lipolysis . these crucial functions of fat , storage , and release of fatty acids are tightly controlled by the key hormones of the fed and fasted states insulin and catecholamines . in addition to these classic functions , the importance of white fat tissue as a central signaling node in systemic metabolism was first identified by the cloning of adipsin and leptin , two important adipokines in fact , fat cells and fat tissues secrete many molecules with crucial roles in metabolism , including tumor necrosis factor ( tnf- ) , adiponectin , resistin , and rbp4 , among others ( rosen and spiegelman , 2014 ) . , defects in adipose differentiation do not lead to healthy , lean animals but instead to lipodystrophy , a serious disease by which other tissues , especially the liver , subsume the function of fat storage , with deleterious effects , including insulin resistance , diabetes , hepatomegaly , and hypertriglyceridemia ( garg , 2011 ) . in contrast to white fat , brown fat is specialized to dissipate chemical energy in the form of heat , defending mammals against hypothermia . it does so by running futile metabolic cycles , most notably the futile cycle of proton exclusion from and leak back into the mitochondrial matrix via the electron transport chain and uncoupling protein 1 ( ucp1 ; reviewed in cohen and spiegelman , 2015 ) . although ucp1 was typically believed to be regulated transcriptionally , a recent study showed that ucp1 can also be regulated posttranslationally , by reactive oxygen species driven sulfenylation of a key cysteine residue ( chouchani , kazak , et al . , 2016 ) . recently a separate futile cycle involving creatine phosphorylation / dephosphorylation was identified in mitochondria of beige fat cells , a type of brown - like adipocyte ( kazak et al . , 2015 ) . of importance , brown fat , in all of its dimensions , plays a role in defending animals against metabolic diseases such as obesity , type 2 diabetes , and hepatic steatosis ( the earliest manifestation of nonalcoholic fatty liver disease [ nafld ] ) . the first evidence in this regard was the observation that mice with genetically ablated ucp1 cells are prone to obesity and diabetes ( lowell et al . , 1993 ) , whereas those with genetically elevated brown fat function are markedly protected from the same disorders ( cederberg et al . , 2001 ) . until recently , the term brown fat was used to refer to ucp1 cells in two distinct anatomical locations : 1 ) developmentally formed depots in the interscapular and perirenal regions , composed mainly of ucp1 adipocytes , which have many small lipid droplets ( termed multilocular ) and dense mitochondria , giving the tissue its characteristic brown color ; and 2 ) ucp1 cells , which are interspersed in many white fat depots , particularly in the subcutaneous regions of rodents and humans . these two types of brown fat are not only distinct cell types ( wu et al . , 2012 ) , but they are also from completely different cell lineages ( seale et al . , 2008 ) . classical brown fat cells , are derived from a skeletal muscle like lineage , as marked by myf5 or pax7 ( seale et al . , 2008 ; lepper and fan , 2010 ) . the beige cells are derived , at least in part , from a vascular smooth muscle like lineage , as marked by the myh11 promoter ( long et al . , 2014 ; most studies have not distinguished between the functional roles of these two types of ucp1 fat cells , as cold exposure or -adrenergic stimulation activates both cell types . recently a murine model has been developed that lacks beige fat cells but has fully functional brown fat ( cohen et al . , 2014 ) . moreover , this obesity occurs exclusively via an excess of subcutaneous fat , a rather unusual finding . these animals have severe hepatic insulin resistance and hepatic steatosis , suggesting that beige fat protects the liver ; whether this occurs through oxidation of circulating lipids by beige cells or through production of a secreted hormone that protects the liver from fat accumulation is not known . an increasing number of factors have been identified that lead to increased ( browning ) or decreased ( whitening ) beige fat activity ( figure 1 ) . depiction of beige adipose tissue , which consists of a mixture of white and beige adipocytes . a schematic of stimuli that lead to increased ( browning ) or decreased ( whitening ) beige fat activity , together with the physiological consequences . adipose tissue was once viewed as a passive repository for triglyceride accumulation within adipocytes but is now appreciated to be a complex tissue containing a host of interacting cell types , including fat cells , immune cells , endothelium , fibroblasts , neurons , and stem cells . although adipocytes account for > 90% of fat pad volume , these other cells types ( collectively referred to as the stromal vascular fraction ) , predominate by overall number ( kanneganti and dixit , 2012 ) . several immune cell subsets are now known to accumulate in adipose tissue and serve important functions . this can be traced back to the observation that adipose tissue produces tnf- and other proinflammatory cytokines , with levels increased in the setting of obesity ; these mediate local and systemic insulin resistance ( hotamisligil et al . , 1993 ) . these cytokines are largely produced by macrophages within the adipose tissue ( weisberg et al . histologically , macrophages can be seen surrounding adipocytes in what have been termed crown - like structures ( cinti et al . , 2005 ) in recent years , the role of immune cell subsets in adipose tissue has become increasingly well understood . in addition to proinflammatory or m1 macrophages , fat also contains alternatively activated or m2 macrophages , with the m1/m2 ratio increasing in obesity ( lumeng et al . , 2007 ) . cold exposure leads to a polarization toward the m2 phenotype , and these m2 cells can produce and secrete catecholamines that stimulate beige fat cells ( nguyen et al . , 2011 ) . eosinophils and type 2 innate lymphoid cells ( ilc2s ) within adipose tissue are also central to beige fat biogenesis . eosinophils produce interleukin ( il)-4 and il-13 , which activate m2 macrophages , and eosinophils themselves can be activated by muscle - derived meteorin - like protein ( qiu et al . ilc2s stimulate beige fat via production of il-33 and enkephalin ( brestoff et al . 2015 ) . regulatory t - cells ( tregs ) are present in visceral adipose tissue but decrease in number with the development of obesity , promoting the development of insulin resistance ( feuerer et al . , 2009 ) . of interest , the properties of visceral fat tregs depend on the expression of peroxisome proliferator - activated receptor ( cipolletta et al . in addition to these immune cell types , roles have also been defined for other t - cell subsets , b - cells , neutrophils , mast cells , and natural killer t - cells ( brestoff and artis , 2015 ) . adipose tissue phenotypes also depend on blood supply and innervation , although the regulation of these processes has been comparatively less studied . as fat mass expands in the setting of overnutrition , local hypoxia can develop , and the oxygen - sensitive transcription factor hypoxia - inducible factor 1 ( hif1 ) can become activated ( krishnan et al . , 2012 ) . genetic and pharmacologic studies show that adipose - specific deletion or inhibition of hif-1 can protect against obesity - related metabolic dysfunction ( jiang et al . , 2011 ; sun et al . , 2013 ) . data also indicate that white and brown adipose tissue can make vascular endothelial growth factor a and other factors to enhance its blood supply ( fredriksson et al . adipose tissue , particularly brown fat , is also extensively innervated with sympathetic fibers that stimulate lipolysis in the setting of fasting , leptin administration , and cold exposure ( bartness et al . , 2010a , b ; zeng et al . , 2015 ) . in contrast brown and beige adipocytes both express high levels of the 3-adrenergic receptor , and pharmacologic activation by cl 316,243 promotes thermogenesis ( himms - hagen et al . , 1994 ) . the factors that regulate the innervation of fat cells remain an area of active investigation . successful targeting of adipose tissue for therapeutic benefit will depend on further clarification of several key unanswered questions . first , what is the full complement of transcriptional regulators that govern the development and maintenance of white , brown , and beige fat ? for example , it is becoming increasingly clear that brown and beige fat do much more than generate heat and may be important endocrine organs ( kajimura et al . , 2015 ) . third , how do different types of fat cells signal to other cell types and tissues , and how do these signals affect systemic metabolism and susceptibility to diabetes , hypertension , cardiovascular disease , and cancer ? finally , can key molecular regulators of adipose tissue be modulated to engineer healthier adipose tissue ? achieving this goal will require a basic understanding of how important factors like prdm16 are physiologically regulated ( e.g. , transcriptionally , translationally , posttranslationally ) . ultimately , any discussion of fat tissues as a target for human therapeutics has to go back to the notion of adipose tissues as the healthiest site for deposition of excess caloric energy ( unger et al . , 2013 ) . we know from human genetics that any inhibition of fat development will cause ectopic lipid deposition and serious disease ( savage et al . , 2003 ) . with that in mind first , with respect to white fat , we might target abnormalities that link the adipose tissues to the consequences of obesity , including diabetes , cardiovascular disorders , and fatty liver disease . as mentioned earlier , adipose tissues in obesity demonstrates aspects of inflammation , including secretion of inflammatory cytokines ; neutralization of cytokines such as tnf improves insulin resistance in rodents ( hotamisligil et al . , similarly , antagonism of the inflammatory protein kinases i - kappa - b kinase epsilon ( ikk ) and tank binding kinase 1 ( tbk1 ) has been shown to improve diabetes in mice ( reilly et al . , 2013 ) . the challenge going forward will be to obtain therapeutic benefit in diabetes or cardiovascular diseases without causing the toxicity associated with generalized suppression of inflammation . for brown and beige fat , the challenge will be to increase their amounts and activities in humans in a safe and effective way . that increased adaptive thermogenesis through brown and beige fat in rodents protects from obesity and diabetes is completely settled science ( cederberg et al . it is also clear that adult humans have substantial stores of beige fat and perhaps some classical brown fat as well ( sharp et al . , 2012 ; wu et al . , 2012 ; cypess et al . , 2013 ; jespersen et al . , 2013 ; lidell et al . , cold exposure or administration of a -3-adrenergic compound have been shown to increase activity of these thermogenic fat depots , as ascertained by fluorodeoxyglucose positron - emission tomographic imaging ( cypess et al . , whether human thermogenic fat can be activated and/or increased in amount to play a strong therapeutic role in diabetes and obesity remains to be seen . several polypeptides , such as fibroblast growth factor 21 ( fgf21 ) and bone morphogenetic protein 7 ( bmp7 ) , can do this in rodents ( tseng et al . , 2008 ; fisher , kleiner , et al . , 2012 ) , but whether the same will be seen in humans with a favorable toxicity profile remains to be seen . additional secreted proteins with thermogenic actions on adipose tissues continue to be discovered ( atrial and ventricular natriuretic peptides and slit2 ; bordicchia et al . , 2012 ; svensson et al . , 2016 ) . it is also worth noting that rodent data cited earlier suggest a hepatoprotective role for beige fat , and so diseases such as nafld may well be the first therapeutic targets for agents that increase beige fat function . the extent to which the diverse metabolic benefits of brown and beige fat are due to enhanced thermogenesis per se or to an endocrine role of these tissues remains an important point to be clarified .

the worldwide epidemic of obesity and type 2 diabetes has greatly increased interest in the biology and physiology of adipose tissues . adipose ( fat ) cells are specialized for the storage of energy in the form of triglycerides , but research in the last few decades has shown that fat cells also play a critical role in sensing and responding to changes in systemic energy balance . white fat cells secrete important hormone - like molecules such as leptin , adiponectin , and adipsin to influence processes such as food intake , insulin sensitivity , and insulin secretion . brown fat , on the other hand , dissipates chemical energy in the form of heat , thereby defending against hypothermia , obesity , and diabetes . it is now appreciated that there are two distinct types of thermogenic fat cells , termed brown and beige adipocytes . in addition to these distinct properties of fat cells , adipocytes exist within adipose tissue , where they are in dynamic communication with immune cells and closely influenced by innervation and blood supply . this review is intended to serve as an introduction to adipose cell biology and to familiarize the reader with how these cell types play a role in metabolic disease and , perhaps , as targets for therapeutic development .

INTRODUCTION TYPES OF FAT CELL BIOLOGY OF ADIPOSE TISSUE UNANSWERED QUESTIONS AND PROSPECTS FOR HUMAN THERAPEUTICS

PMC4333621

quercus infectoria olivier galls [ figure 1 ] were purchased from the local herbal shop in kota bharu , kelantan and identified based on their physical appearances which were globular in shape , 0.8 cm to 2.5 cm in diameter , green - yellow in color , odor is slight , strongly pungent taste and tuberculated surface . the galls were washed with distilled water , left dry at room temperature before they were crushed and ground prior to the extraction . gall of quercus infectoria olivier the methanol extract was prepared by immersing 100 g of the q. infectoria gall powder in 500 ml of absolute methanol ( merck ) for 72 h in 50c water bath . the filtrates were concentrated under reduced pressure using a rotary evaporator at temperature of 55c . the resulting pellet was finally pounded to dryness at 50c for 48 h to produce a powdered and brown crude extract . the aqueous extract was prepared by immersing 100 g of q. infectoria powder in gall 500 ml of sterile distilled water for 72 h in 50c water bath . the mixture was then prefiltered using a coffee filter and then filtered using whatmann filter paper no 1 . the filtrates were concentrated under reduced pressure using a rotary evaporator at a temperature of 80c . the resulting pellet was freeze - dried at 50c under vacuum until the pellet produce a fine crystal - like crude extract . the extracts were dissolved in sterile distilled water to a final concentration of 100 mg / ml for disc diffusion technique and 64 mg / ml for broth microdilution technique . blank discs ( oxoid ) were impregnated with the desired volume of extract solutions to get the final concentration of 1.0 , 2.0 and 5.0 mg / disc and allowed to dry in sterile condition . five american type culture collection ( atcc ) strains of candida species were used in this study ; c. albicans atcc 10231 , c. tropicalis atcc 13803 , c. parapsilosis atcc 20019 , c. glabrata atcc 90525 and c. krusei atcc 6258 . similarly , one archived clinical isolates was selected for each candida species obtained from hospital universiti sains malaysia ( husm ) kubang kerian , kelantan . yeasts were subcultured and maintained onto sabouraud dextrose agar ( oxoid ) and potato dextrose agar ( difco ) at 35c for 24 h. the yeast suspension of each strain was prepared at a concentration of 10 cells / ml or mcfarland equivalent of 0.5 for disc diffusion test and broth dilution assay . the procedures described here for disc diffusion test and determination of minimum inhibitory concentration ( mic ) values are in accordance with standard international recommendations provided by the clinical and laboratory standards institute . the standardized test inoculum was spread in three directions onto the surface of the mueller hinton agar using a sterile cotton swab . extract discs were placed on the inoculated agar surface along with negative and positive control within 15 min of inoculation . disc impregnated with sterile distilled water was used as negative control , while amphotericin b disc ( 10 g ) was used as positive control . all plates were incubated at 35c for 24 h. the anti - candida activity was observed from the size of the inhibition zone diameter surrounding the disc measured in millimeters ( mm ) . the mic values of each extract against the candida strains were determined using a twofold serial microdilution of extracts with concentration ranging from 16 mg / ml to 0.03 mg / ml . equal volumes of diluted inoculums suspensions were added to the designated test and control wells . the mic values were taken as the lowest concentrations of extracts showing no turbidity after 24 h incubation at 35c . the wells with absence turbidity were subcultured onto sabouraud dextrose agar and incubated at 35c for 24 h to determine the minimum fungicidal concentration ( mfc ) values . the phytochemical qualitative tests were carried out for both methanol and aqueous extracts of q. infectoria to screen for the presence of tannin , saponin , phenol , flavonoids and alkaloid using standard procedure . the gas chromatography ( gc ) system ( agilent 7890a ) was equipped with triple axis detector ( agilent 5975c ) and an autosampler ( agilent 7693 ) . the column temperature was programmed from an initial temperature of 70c to a final temperature of 280c with increased time at the 20c / min . the injector temperature was 280c and injection volume was 5 l . the carrier gas was helium ( 1 ml / min ) . identification of phytochemical components was performed by diluting 1 gram of crude extracts into 5 ml of appropriate solvents . data entry and statistical analysis were performed using ibm corp . released for windows : ibm spss software version 20 . student 's t - test was used for statistical comparison and p < 0.05 were considered as significant . quercus infectoria olivier galls [ figure 1 ] were purchased from the local herbal shop in kota bharu , kelantan and identified based on their physical appearances which were globular in shape , 0.8 cm to 2.5 cm in diameter , green - yellow in color , odor is slight , strongly pungent taste and tuberculated surface . the galls were washed with distilled water , left dry at room temperature before they were crushed and ground prior to the extraction . the methanol extract was prepared by immersing 100 g of the q. infectoria gall powder in 500 ml of absolute methanol ( merck ) for 72 h in 50c water bath . the filtrates were concentrated under reduced pressure using a rotary evaporator at temperature of 55c . the resulting pellet was finally pounded to dryness at 50c for 48 h to produce a powdered and brown crude extract . the aqueous extract was prepared by immersing 100 g of q. infectoria powder in gall 500 ml of sterile distilled water for 72 h in 50c water bath . the mixture was then prefiltered using a coffee filter and then filtered using whatmann filter paper no 1 . the filtrates were concentrated under reduced pressure using a rotary evaporator at a temperature of 80c . the resulting pellet was freeze - dried at 50c under vacuum until the pellet produce a fine crystal - like crude extract . the extracts were dissolved in sterile distilled water to a final concentration of 100 mg / ml for disc diffusion technique and 64 mg / ml for broth microdilution technique . blank discs ( oxoid ) were impregnated with the desired volume of extract solutions to get the final concentration of 1.0 , 2.0 and 5.0 mg / disc and allowed to dry in sterile condition . five american type culture collection ( atcc ) strains of candida species were used in this study ; c. albicans atcc 10231 , c. tropicalis atcc 13803 , c. parapsilosis atcc 20019 , c. glabrata atcc 90525 and c. krusei atcc 6258 . similarly , one archived clinical isolates was selected for each candida species obtained from hospital universiti sains malaysia ( husm ) kubang kerian , kelantan . yeasts were subcultured and maintained onto sabouraud dextrose agar ( oxoid ) and potato dextrose agar ( difco ) at 35c for 24 h. the yeast suspension of each strain was prepared at a concentration of 10 cells / ml or mcfarland equivalent of 0.5 for disc diffusion test and broth dilution assay . the procedures described here for disc diffusion test and determination of minimum inhibitory concentration ( mic ) values are in accordance with standard international recommendations provided by the clinical and laboratory standards institute . the standardized test inoculum was spread in three directions onto the surface of the mueller hinton agar using a sterile cotton swab . extract discs were placed on the inoculated agar surface along with negative and positive control within 15 min of inoculation . disc impregnated with sterile distilled water was used as negative control , while amphotericin b disc ( 10 g ) was used as positive control . all plates were incubated at 35c for 24 h. the anti - candida activity was observed from the size of the inhibition zone diameter surrounding the disc measured in millimeters ( mm ) . the mic values of each extract against the candida strains were determined using a twofold serial microdilution of extracts with concentration ranging from 16 mg / ml to 0.03 mg / ml . equal volumes of diluted inoculums suspensions were added to the designated test and control wells . the mic values were taken as the lowest concentrations of extracts showing no turbidity after 24 h incubation at 35c . the wells with absence turbidity were subcultured onto sabouraud dextrose agar and incubated at 35c for 24 h to determine the minimum fungicidal concentration ( mfc ) values . the standardized test inoculum was spread in three directions onto the surface of the mueller hinton agar using a sterile cotton swab . extract discs were placed on the inoculated agar surface along with negative and positive control within 15 min of inoculation . disc impregnated with sterile distilled water was used as negative control , while amphotericin b disc ( 10 g ) was used as positive control . all plates were incubated at 35c for 24 h. the anti - candida activity was observed from the size of the inhibition zone diameter surrounding the disc measured in millimeters ( mm ) . the mic values of each extract against the candida strains were determined using a twofold serial microdilution of extracts with concentration ranging from 16 mg / ml to 0.03 mg / ml . equal volumes of diluted inoculums suspensions were added to the designated test and control wells . the mic values were taken as the lowest concentrations of extracts showing no turbidity after 24 h incubation at 35c . the wells with absence turbidity were subcultured onto sabouraud dextrose agar and incubated at 35c for 24 h to determine the minimum fungicidal concentration ( mfc ) values . the phytochemical qualitative tests were carried out for both methanol and aqueous extracts of q. infectoria to screen for the presence of tannin , saponin , phenol , flavonoids and alkaloid using standard procedure . the gas chromatography ( gc ) system ( agilent 7890a ) was equipped with triple axis detector ( agilent 5975c ) and an autosampler ( agilent 7693 ) . the column temperature was programmed from an initial temperature of 70c to a final temperature of 280c with increased time at the 20c / min . the injector temperature was 280c and injection volume was 5 l . the carrier gas was helium ( 1 ml / min ) . identification of phytochemical components was performed by diluting 1 gram of crude extracts into 5 ml of appropriate solvents . data entry and statistical analysis were performed using ibm corp . released for windows : ibm spss software version 20 . student 's t - test was used for statistical comparison and p < 0.05 were considered as significant . screening of methanol and aqueous extracts against all candida species exerted an increasing mean inhibition zone diameter in a concentration dependent manner . at lower concentrations ( 1.0 mg / disc and 2.0 mg / disc ) , the extracts inhibited the growth of yeasts weakly ( between 7 and 10 mm in diameter ) or not at all . both extracts inhibited all candida species with some strains exhibited greater inhibition zone diameter compared to the positive control at concentration of 5.0 mg / disc [ table 1 ] . the lowest mic and mfc values detected were 0.06 mg / ml as shown in table 2 . both methanol and aqueous extracts showed relatively similar anti - candida activity . c. krusei appeared the most susceptible candida species showing low mic and mfc values . anti - candida activity of q. infectoria gall extracts against candida species by disc diffusion test mic and mfc values in mg / ml of q. infectoria gall extracts against candida species preliminary phytochemical screening was performed to establish the profile of gall extracts for its chemical composition . both methanol and aqueous q. infectoria gall extracts showed the presence of tannins [ table 3 ] . the result of gc - mass spectrometry ( gc - ms ) analysis of the crude extracts of q. infectoria revealed two major compounds scanned by the nisto5a.l database . the active principle , retention time ( rt ) , molecular weight and molecular structure were presented in figure 2 . 1 , 2 , 3 - benzenetriol ( pyrogallol ) in both methanol and aqueous extracts of q. infectoria gall was identified in high percentage , which accounted for 81.66% and 100% of the total respectively . phytochemical screening of q. infectoria gall extracts chromatogram of methanol extract ( a ) and aquoeus extract ( b ) of quercus infectoria gall by gas chromatography - mass spectrometry ( gc - ms ) analysis . the gc - ms spectrum at retention time 14.50 min of major compound of the 1,2,3-benzenetriol ( pyrogallol ) with molecular formula of c6h6o3 and molecular weight of 126.0 g / mol ( c ) candida albicans has historically been the predominant species causing candida infections . however , the emergence of nac with intrinsic resistance to azoles represents a major challenge for empirical , therapeutic and prophylactic strategies in the near future especially in immunocompromised and severely ill - patients . the most common nac species causing candidiasis include c. glabrata , c. tropicalis , c. parapsilosis and c. krusei . selection of candida species in this study were based on previous reported candida isolates identified in various spectra of candidiasis in husm . the results obtained from this study were in agreement with the previous studies on antibacterial acitivities of q. infectoria gall extracts . in our study , the gall extracts exhibited a substantial in - vitro anti - candida activity against all tested candida species . in general , both methanolic and aqueous extracts effectively inhibited the growth of the yeasts at the minimum concentration of 5 mg / disc , which is slightly higher than the concentrations of disc extracts used to screen anti - bacterial activity ( 1 mg / disc or 2 mg / disc ) by disc diffusion test . in this study , we obtained relatively similar mic values for both atcc and clinical isolates of all candida species in either methanolic or aqueous extract . lowest mic values were observed for both extracts against isolates of c. krusei when compared to other candida species . lower mic values were also seen for c. glabrata . c. krusei is intrinsically resistant to fluconazole and demonstrates decreased susceptibility to amphotericin b , thus requiring higher doses to be used for treatment . minimum fungicidal concentrations are one of various in vitro microbiological parameters used to determine the fungicidal activity of antimicrobial agents . microbiological definition of bactericidal or fungicidal activity has been taken arbitrarily as a ratio of mfc to mic of 4 or less . in this study , the mfc / mic ratio of aqueous extract against c. parapsilosis , clinical isolates of c. krusei and c. glabrata were > 4 , which were considered fungistatic . fungicidal activities were observed in the methanol extract against almost all tested candida species . the constant increase in the number of immunocompromised individuals has resulted in an ever - growing number of serious fungal infections due to c. albicans and c. krusei . most serious fungal infections occur in profoundly immunosuppressed individuals , thus it is generally assumed that a cidal activity would be preferable . however , a clear advantage of fungicidal activity over fungistatic activity remains elusive . some example shows that despite being a fungistatic drug , the clinical trials shows effective result in treating candidiasis . a further study required to determine whether there are clinical settings in which a fungicidal drug may provide an improved response when compared with a fungistatic drug in the treatment of fungal infection . the introduction of active antifungal agents with increased potency action offers new hope for improved therapeutic outcomes and has resulted in a need to assess the fungicidal activity of these agents . a different organic solvent used as however , this characteristic is also relying on the polarity of the solvent and compound to be extracted . some hydrolysable tannins have shown to be more reactive and have stronger inhibitory effects than condensed tannins . pyrogallol , one of hydrolysable tannin with high molecular weight and glucose esters of phenolic acids was the main bioactive constituent found from both methanol and aqueous extracts in our study . the presence of hydroxyl groups and alpha - beta double bonds in a phenolic compound [ figure 3 ] play an important role toward antimicrobial activity . pyrogallol has been reported to have various biological activities such as candidicidal and fungicidal activities . those activities were possibly triggered by the presence of three hydroxyl groups in the structure , which eventually affects the biosynthesis of cell wall and cell membrane . changes in the permeability of cell membrane could cause a decrease in cell volume , thus abnormalities may attribute to cell membrane alterations . chemical structure of pyrogallol ( 1 , 2 , 3 benzenetriol ) pyrogallol is the extracted compound that potentially possesses anti - candida properties , which can be further explored for detail evaluations in near future . however , the use of isolated bioactive compound in elucidating maximum antimicrobial activity is doubtful because it has been found that the whole herbal extracts are more effective than isolated phytochemicals due to synergistic effect between the phytochemical components . therefore , whole extract formulation with a standard marker of bioactive compound is a need in order to make use the synergistic effect of existing compounds . it is intriguing to note that crude extracts of q. infectoria galls hold an anti - candida potential , which might be further explored to be used as an alternative for treatment and control of some fungal infections . further investigations are needed to develop a standardized q. infectoria gall extract and to understand its mechanism of anti - candida activity .

background : galls of quercus infectoria have been traditionally used to treat common ailments , including yeast infections caused by candida species.objective:this study aimed to evaluate the in vitro anti - candida activity of q. infectoria gall extracts against selected candida species.materials and methods : methanol and aqueous extracts of q. infectoria galls were tested for anti - candida activity against candida albicans , candida krusei , candida glabrata , candida parapsilosis and candida tropicalis . the minimum inhibitory concentrations were determined using the two - fold serial dilution technique of concentrations ranging from 16 mg / ml to 0.03 mg / ml . after 24 h , the minimum fungicidal concentrations were determined by subculturing the wells , which showed no turbidity on the agar plate . potential phytochemical group in the crude extracts was screened by phytochemical qualitative tests and subsequently subjected to the gas chromatography - mass spectrometry analysis.results:both methanol and aqueous extracts displayed substantial anti - candida activity and pyrogallol was the major component of both crude extracts.conclusions:data from current study suggested that q. infectoria gall extracts are a potential source to be developed as anti - candidiasis .

Materials and Methods Plant material Preparation of extracts Microorganisms and preparation of inoculum Anti- Screening by disc-diffusion test Determination of minimum inhibitory concentration and minimum fungicidal concentrations values Phytochemical analysis Gas chromatography-mass spectrometry analysis Statistical analysis Results Discussion Conclusion

PMC4738917

physiological responses to repeated sprint exercise ( rse ) and recovery periods are important topics of study for the purpose of developing effective strategies to improve sport performance . with increased participation of women in team sports , understanding gender differences in rse is a necessary consideration . the wingate test has been used to test gender differences in anaerobic performance characterized by peak and mean power outputs ( 12 , 13 , 16 , 18 , 22 , 26 , 30 ) . women achieve approximately 60% of peak power output of men ; however , body mass accounts for much of this difference ( 26 , 30 ) . differences in metabolism between men and women performing a single wingate test have been demonstrated with lower lactate accumulation and use of type i fiber glycogen in women ( 12 , 16 ) . additionally , the relative contribution of the aerobic energy system may be higher in women , possibly due to less reliance on glycolytic processes ( 12 , 14 , 19 , 27 ) . specific to rse performance , a decrease in power output from one sprint to the next has been demonstrated in men but not in women during repeated wingate tests ( 11 ) . likewise , when repeated explosive strength or resistance exercises are performed , women demonstrate a slower rate of fatigue and faster acute recovery ( 14 , 17 , 20 , 27 ) . these types of exercises have been useful in describing anaerobic performance differences between men and women ; however , long duration ( 30 sec ) sprints and resistance exercise do not accurately simulate the repeated short duration ( < 10 sec ) sprints performed during many types of sports . thus , observing gender differences during repeated short duration sprints may be more applicable to the athlete . few studies have addressed gender differences in this area and results are somewhat conflicting ( 2 , 9 , 20 , 32 ) . ( 2 ) observed greater decline in power output during an 8-sec cycling sprint in women compared to men ; however women were capable of recovering more power output for a second sprint following a 4- min recovery period . during ten 6-sec running sprints , brooks et al . ( 9 ) observed similar decreases in power output in men and women . yet , in another study a greater drop in running sprint performance was noted in boys compared to girls performing ten 5-sec sprints ( 32 ) . similar results have been demonstrated in adult men and women performing repeated 30-m sprints ( 20 ) . overall , physiological differences between men and women in response to rse are not clearly defined . based on the limited data concerning gender differences , evidence suggests that women rely less on glycolytic processes and more on aerobic metabolism during repeated high intensity exercise ( 11 , 14 , 19 , 27 ) . aerobic fitness of men and women appears to be related to rse performance in that those with higher maximal oxygen uptake ( vo2max ) or lactate threshold demonstrate less decline in power output ( 4 , 6 , 7 , 23 , 29 ) . thus , an important consideration when investigating gender differences during rse is aerobic fitness and training background . because men typically have higher vo2max and greater fat free mass ( ffm ) , matching men and women for vo2max expressed relative to ffm helps to identify males and females with similar aerobic capacities and thus , may provide some clarity to gender differences during rse . therefore , the purpose of this study was to examine gender differences in rse performance among male and female athletes matched for vo2max relative to ffm ( vo2max ffm ) . we hypothesized that women would demonstrate less of a decline in power output during short repeated sprints and a greater recovery of power output following 5 min of active recovery . thirty - nine college athletes ( 17 males and 22 females ) were recruited from division ii university sport teams . participants represented men s soccer , women s soccer , baseball , softball , women s basketball and volleyball . each participant read and signed an informed consent form approved by the university s institutional review board . all participants were free of injury or illness and at low risk for a cardiovascular event during exercise . hydrostatic weight of each participant was obtained via hydrostatic weighing to determine percent body fat and ffm . measurements were taken with a calibrated chatillon 806h mechanical scale ( precision weighing balances , hartford , ma ) read to the nearest 0.1 kg while the participant was completely submerged underwater and had blown out as much air as possible . repeated measures were taken until there was no further increase in hydrostatic weight and at least 5 measurements had been obtained . the highest three measures were averaged and recorded as the participant s hydrostatic weight . body density was estimated by correcting for residual volume and gas trapped in the gi tract , which was assumed to be 100 ml ( 31 ) . the siri equation ( 28 ) was used to convert body density to percent body fat of each participant . ffm was calculated as body mass ( 1- the fraction of body fat ) . following the hydrostatic weighing , the participant completed a gxt on a motorized treadmill ( quinton , medtrack sr60 , bothell , wa ) for measurement of vo2max . prior to each test , flow volume and gas ( co2 and o2 ) calibrations were performed on an automated gas analyses system ( parvomedics trueone 2400 , sandy , ut ) . a 16% o2 and 4% co2 gas mixture the gxt protocol was continuous and included 2-min stages performed at a running speed determined during a 5-min warm - up period and associated with a rate of perceived exertion of 1213 on the borg scale ( 8) . during the test , running speed was maintained while incline was increased 2.5% at each stage until volitional exhaustion . vigorous prompts and encouragement were provided , especially during the final minutes . throughout the gxt oxygen uptake was measured continuously from the collection and analyses of expired air . vo2max was determined from the average of single - breath data collected during the final 30 sec . vo2 ( mlmin ) was divided by ffm ( kg ) to determine vo2max ffm . eleven pairs of males and females matched for vo2max ffm were scheduled to complete a rse protocol . prior to performing the rse protocol , the men and women paired for vo2max ffm completed a questionnaire that included daily dietary , sleep and training habits . from this , we concluded that the participants did not vary remarkably in schedules and habits ( all athletes lived on the university campus and consumed daily meals from the student cafeteria ) . further , it did not appear that any athlete was attempting to lose weight through dieting or excessive exercise . in preparation for the rse , each participant was provided an energy bar and nutrition supplement drink ( 480 calories total ) to consume during the 24-hr period prior to the rse . instructions covering the two days prior to the rse included the following ; consume meals regularly , consume the additional 480 calories the day before the test , drink extra fluids , avoid alcohol at least 24-hr prior , consume a meal and extra fluids 23 hrs prior , and get adequate sleep the night before the test . participants were also instructed to consume additional carbohydrates ( a list of carbohydrate - rich foods typically available to the participants was provided ) the evening before and the morning of the rse . body weight was measured daily for five days prior to the rse at the same time each day . day - to - day fluctuations did not exceed 2% of original body weight and all but two participants began the rse at a higher body weight . rse was scheduled so that the participant did not engage in strenuous physical activity during the 24-hr period prior to the test . all participants reported that instructions were followed and were able to consume additional calories during the two days prior to the rse . a monark ergomedic 894e peak cycle ( healthcare international , seattle wa ) was used for the rse protocol . the protocol consisted of a 5-minute warm - up that included three short maximal accelerations . the average absolute power output ( po , watts ) achieved during each of the 6-sec sprints was determined . peak power output ( ppo ) was calculated from the initial 5 sec and the final power output ( fpo ) was calculated from the final 5 sec during the 30-sec sprint . power output was expressed in absolute terms and relative to ffm ( poffm , wattkg ffm ) . for vo2 measures , vo2 measures were expressed relative to ffm ( vo2ffm ) and determined from the average of the single breath data during each 6-sec sprint , the 5-min recovery period , and the initial and final 5 seconds of the 30-sec sprint . the five 6-sec cycle sprints were analyzed using a two - way repeatedmeasures anova ( sprint gender ) . in the case of a significant sprint effect , a bonferroni test for multiple comparisons was performed and significance readjusted to p .01 ( .05/5 ) . two - way repeated measures were also performed to test differences between the initial 5 sec and final 5 sec of the 30-sec sprint and the interactive effect between gender and time . for descriptive characteristics , a paired ttest was used to compare genders . analyses of data were completed using the statistical package for the social sciences , version 17 ( spss inc , chicago , il ) . thirty - nine college athletes ( 17 males and 22 females ) were recruited from division ii university sport teams . participants represented men s soccer , women s soccer , baseball , softball , women s basketball and volleyball . each participant read and signed an informed consent form approved by the university s institutional review board . all participants were free of injury or illness and at low risk for a cardiovascular event during exercise . hydrostatic weight of each participant was obtained via hydrostatic weighing to determine percent body fat and ffm . measurements were taken with a calibrated chatillon 806h mechanical scale ( precision weighing balances , hartford , ma ) read to the nearest 0.1 kg while the participant was completely submerged underwater and had blown out as much air as possible . repeated measures were taken until there was no further increase in hydrostatic weight and at least 5 measurements had been obtained . body density was estimated by correcting for residual volume and gas trapped in the gi tract , which was assumed to be 100 ml ( 31 ) . the siri equation ( 28 ) was used to convert body density to percent body fat of each participant . ffm was calculated as body mass ( 1- the fraction of body fat ) . following the hydrostatic weighing , the participant completed a gxt on a motorized treadmill ( quinton , medtrack sr60 , bothell , wa ) for measurement of vo2max . prior to each test , flow volume and gas ( co2 and o2 ) calibrations were performed on an automated gas analyses system ( parvomedics trueone 2400 , sandy , ut ) . a 16% o2 and 4% co2 gas mixture the gxt protocol was continuous and included 2-min stages performed at a running speed determined during a 5-min warm - up period and associated with a rate of perceived exertion of 1213 on the borg scale ( 8) . during the test , running speed was maintained while incline was increased 2.5% at each stage until volitional exhaustion . vigorous prompts and encouragement were provided , especially during the final minutes . throughout the gxt oxygen uptake was measured continuously from the collection and analyses of expired air . vo2max was determined from the average of single - breath data collected during the final 30 sec . vo2 ( mlmin ) was divided by ffm ( kg ) to determine vo2max ffm . eleven pairs of males and females matched for vo2max ffm were scheduled to complete a rse protocol . prior to performing the rse protocol , the men and women paired for vo2max ffm completed a questionnaire that included daily dietary , sleep and training habits . from this , we concluded that the participants did not vary remarkably in schedules and habits ( all athletes lived on the university campus and consumed daily meals from the student cafeteria ) . further , it did not appear that any athlete was attempting to lose weight through dieting or excessive exercise . in preparation for the rse , each participant was provided an energy bar and nutrition supplement drink ( 480 calories total ) to consume during the 24-hr period prior to the rse . instructions covering the two days prior to the rse included the following ; consume meals regularly , consume the additional 480 calories the day before the test , drink extra fluids , avoid alcohol at least 24-hr prior , consume a meal and extra fluids 23 hrs prior , and get adequate sleep the night before the test . participants were also instructed to consume additional carbohydrates ( a list of carbohydrate - rich foods typically available to the participants was provided ) the evening before and the morning of the rse . body weight was measured daily for five days prior to the rse at the same time each day . day - to - day fluctuations did not exceed 2% of original body weight and all but two participants began the rse at a higher body weight . rse was scheduled so that the participant did not engage in strenuous physical activity during the 24-hr period prior to the test . all participants reported that instructions were followed and were able to consume additional calories during the two days prior to the rse . a monark ergomedic 894e peak cycle ( healthcare international , seattle wa ) was used for the rse protocol . the protocol consisted of a 5-minute warm - up that included three short maximal accelerations . the average absolute power output ( po , watts ) achieved during each of the 6-sec sprints was determined . peak power output ( ppo ) was calculated from the initial 5 sec and the final power output ( fpo ) was calculated from the final 5 sec during the 30-sec sprint . power output was expressed in absolute terms and relative to ffm ( poffm , wattkg ffm ) . for vo2 measures , vo2 measures were expressed relative to ffm ( vo2ffm ) and determined from the average of the single breath data during each 6-sec sprint , the 5-min recovery period , and the initial and final 5 seconds of the 30-sec sprint . the five 6-sec cycle sprints were analyzed using a two - way repeatedmeasures anova ( sprint gender ) . in the case of a significant sprint effect , a bonferroni test for multiple comparisons was performed and significance readjusted to p .01 ( .05/5 ) . two - way repeated measures were also performed to test differences between the initial 5 sec and final 5 sec of the 30-sec sprint and the interactive effect between gender and time . for descriptive characteristics , analyses of data were completed using the statistical package for the social sciences , version 17 ( spss inc , chicago , il ) . of the 39 participants tested , 11 male ( 6 baseball and 5 soccer athletes ) and female ( 5 soccer , 4 softball , 1 volleyball and 1 basketball athlete ) pairs were identified based on vo2max relative to ffm . percent difference in vo2max ffm between matched pairs ranged from 0.1 to 2.6% ( mean sd , 1.0 0.8% ) . men achieved higher absolute po than women during each of the five sprints and at the beginning and end of the 30-sec sprint ( table 2 ) . the main effect of 6-sec sprints was a decline in po but multiple comparison statistics revealed this to occur only between sprint 2 and each of the final two sprints . fpo at the end of the 30-sec sprint was lower than ppo for both men and women . no gender differences in poffm during the five 6-sec sprints were observed ( figure 1 ) . during the 30-sec sprint , ppoffm was higher in women while no difference in fpoffm between men and women was seen . ppoffm was greater ( p .05 ) than fpoffm in both men and women and an interactive effect between poffm and gender revealed a greater ( p .05 ) decline in poffm in women during the 30sec sprint . during the five 6-sec sprints , vo2ffm increased from sprint 1 to sprint 2 , with no further increases observed thereafter ( figure 2 ) . vo2ffm did not differ between men and women during the 6-sec sprints . during the 5-min recovery period , vo2ffm increased during the 30-sec sprint in both men and women ( p .05 ) . we tested repeated sprint performance in male and female college athletes matched for vo2max relative to ffm and observed no difference between genders in the ability to maintain power output during five 6-sec cycle sprints . a previous report also found a similar decline in power output over 10 sprints between men and women despite the greater work output in men ( 9 ) . however , gender differences have been reported elsewhere ( 2 , 11 , 32 ) . in one study , women demonstrated greater decline in power output during an 8-sec sprint , but recovered more power output for the second sprint compared to men ( 2 ) . during three 30-sec sprints , esbjrnsson - liljedahl et al . ( 11 ) observed a significant decrease in power output from sprint 1 in men , but not in women . further , a greater power output decline in males has been shown during repeated running sprints ( 20 , 32 ) . it is possible that inadequate matching of men and women for aerobic capacity could have contributed to the observed differences reported in these studies . likewise , the lack of gender difference in this study regarding power output loss during short sprints may be attributed to the similar aerobic capacity between men and women . in this study , men and women actively recovered for five minutes following the five 6-sec sprints before an all - out 30-sec sprint was performed . we observed a greater recovery of poffm in women at the beginning of the 30-sec sprint compared to men . a higher rate of recovery following high intensity exercise in women has been previously demonstrated ( 2 , 14 , 17 ) . as noted earlier , billaut et al . ( 2 ) observed greater recovery of power output when a second 8-sec sprint was performed following a short recovery period . likewise , faster acute recovery from intermittent isometric exercise performed at maximal voluntary contraction has been demonstrated in women ( 14 , 17 ) . because recovery during repeated sprints relies heavily on aerobic processes ( 1 , 5 , 7 , 15 ) , the greater recovery of po demonstrated in women in this study may be associated with a greater aerobic contribution to recovery , evidenced from the higher vo2ffm observed in women . in addition to having a higher ppoffm , women in this study demonstrated a greater absolute drop in power output during the 30-sec sprint . contrary to this , the decrease in power output during the wingate test has been reported to be lower in women ( 13 ) . however , the greater drop observed in men may have been related to their higher initial po because when change in power output is expressed as a relative change ( ( ppo - fpo)/ppo 100 ) , the differences disappear ( 13 , 18 , 30 ) . thirty - second sprint performance following four minutes of recovery is characterized by an increased reliance on aerobic energy processes associated with phosphocreatine depletion and a reduction in glycolytic rate ( 7 ) . as an indication of this , we observed an increase in vo2 in both men and women during the sprint . however , vo2 was higher in women than men , despite the greater drop in power output . evidence that women rely more on oxidative phosphorylation compared to men during high intensity exercise has been shown ( 19 , 27 ) . further evidence for this is the lower rate of glycogen use observed in women during repeated 30-sec sprints ( 11 ) . thus , it is possible that women do not draw from glycolytic processes enough to maintain power output and may rely more heavily on aerobic processes ( 11 , 14 , 19 , 27 ) . a limitation to this study is that vo2max was determined from a treadmill gxt , while repeated sprint performance was measured during cycling . thus , it is possible that men and women were not matched for cycle vo2max relative to ffm . another limitation is that diet intake was not controlled prior to rse ; thus it can not be accurately determined if athletes maintained adequate intake . however , given that body weight change was minimal for five days prior to the rse and that most athletes demonstrated an increase in body weight , we believe this provides some evidence that the athletes received adequate hydration and energy intake prior to the rse protocol . in summary , when matched for vo2max relative to ffm , male and female college athletes achieve similar poffm during five 6-sec sprints separated by 30 seconds of active recovery . during an active 5-min recovery women were better able to recover power output at the beginning of a 30-sec sprint , but then demonstrated a greater drop in po despite having higher vo2ffm at the beginning and end of the sprint . these data indicate that men and women with similar aerobic capacities do not respond differently to short repeated sprints but may differ in their ability to recover and perform sprints of longer duration . our results contribute to the limited data concerning gender differences in sprint performance ; however , further research directed toward the physiological mechanisms associated with faster recovery in women is needed . addressing this issue with a training study that includes men and women initially matched for aerobic and anaerobic powers might add significant insight into the gender differences observed during repeated sprints . women s increased participation in team sports raises the importance of learning how men and women differ in response to exercise in order to develop effective training strategies for each gender . regarding current training practices , our data suggest that male athletes may benefit by shifting the focus somewhat away from peak power improvements and toward enhancing aerobic endurance or ability to recovery from repeated sprints . female athletes may benefit from an increased focus on anaerobic endurance or ability to sustain high power output for long durations .

the purpose of this study was to examine gender differences in repeated sprint exercise ( rse ) performance among male and female athletes matched for vo2max relative to ffm ( vo2max ffm ) . thirty nine male and female college athletes performed a graded exercise test for vo2max and hydrostatic weighing to determine ffm . from the results , 11 pairs of males and females matched for vo2max ffm ( mean sd ; 58.3 4.3 and 58.9 4.6 mlkg ffm1min1 ; men and women , respectively ) were identified . on a separate day , matched participants performed a rse protocol that consisted of five 6-sec cycle sprints with 30-sec recovery periods , followed by 5-min active recovery and a 30-sec all - out sprint . repeated 6-sec sprint performance did not differ between men and women ; both maintained power output ( po ) until sprint 4 . poffm ( wkg1 ffm ) did not differ between men and women during the five sprints . during the 30-sec sprint , men achieved a lower peak poffm than women ( 11.7 1.5 vs 13.2 1.2 ) ; however , the decline in poffm over 30 sec was greater in women . vo2 ( mlkg ffm1min1 ) was lower in men during recovery ( 24.4 3.8 vs 28.7 5.7 ) and at the beginning ( 29.2 4.0 vs 34.7 4.9 ) and end ( 49.4 5.0 vs 52.3 4.0 ) . of the 30-sec sprint . these data indicate that men and women with similar aerobic capacities do not respond differently to short repeated sprints but may differ in their ability to recover and perform sprints of longer duration .

INTRODUCTION METHODS Participants Protocol Repeated Sprint Exercise Statistical Analysis RESULTS DISCUSSION

PMC3480029

traditional cardiac surgery requires a sternotomy , cardiopulmonary bypass , and cardiac arrest to provide a still and bloodless heart and its vessels for operation . while necessary , these interventions are invasive and traumatic . the morbidity of cardiac surgery can be quite a burden to the patients [ 13 ] . in order to reduce the risks associated with open - heart surgery , cardiac surgeons operate through small incisions in the chest , eliminating the need for a sternotomy , stopping the heart , or requiring a heart - lung machine to be used . decreased trauma to tissue and muscle with smaller incisions typically results in less pain . avoiding the bypass machine minimally invasive cardiac surgery , in comparison to traditional procedures , offers many benefits including reduction of the chance for postsurgical complications and leads to shorter hospital stay with a faster return to normal activities . aortic valve replacement is such a cardiac procedure that can be performed with minimally invasive techniques . in the last decade , transcatheter aortic valve replacement ( tavr ) has been studied for treating the patients of high surgical risk . the bioprosthetic valves are delivered through catheters transfemorally [ 813 ] or transapically [ 1418 ] and are implanted within the diseased aortic valve . in current clinical practice , the transfemoral approach is the first choice , while the transapical method is only chosen for patients who have poor vascular access . however , the transapical aortic valve approach may be more applicable to a wider range of patients because of the lack of physical anatomic limitations . antegrade access avoids possible complication with retrograde access , which is caused by inability to cross a stenotic valve . larger sheath diameters used in the transapical access lead to less need for crimping of the valves , which may be translated into better prosthesis longevity [ 20 , 21 ] . early , midterm , clinical , and echocardiographic outcomes indicate that both approaches are comparable , despite a significantly higher risk profile in the cohort treated with the transapical approach . typically , the imaging employed for tavr is primarily high - resolution fluoroscopy and adjunctive 2-dimensional m - mode transesophageal echocardiography . the problems with fluoroscopy guidance include device embolization , coronary obstruction , low or high placement , misalignment , landmark loss ( after ballooning the valve the calcium pattern used by fluoroscopy to identify the leaflets / annulus is changed ) , perivalvular leaks , need for rapid ventricular pacing , radiation exposure , and intravenous contrast toxicities . all of these are imaging related and may be improved with better imaging ; hence our desire is to pursue magnetic resonance imaging guidance . mri provides excellent visualization particularly in its ability to provide high - resolution images of blood - filled structures without additional risk of radiation or contrast reaction . new generations of open , wide , and short bore mr scanners and real - time sequences made mri not only cardiovascular diagnostics but also minimally invasive cardiac surgery possible . the confined physical space of the mri scanner , even with a wider and shorter bore , can be a challenging environment in which valve replacement is performed . during the procedure , the surgeon must manipulate the different components of the delivery device and other tools through the delivery device while visualizing the in - room mri display simultaneously . in order to deliver the prosthesis properly , a coordinated effort between the surgeon and the team is critical in the noisy mri environment while contending with respiratory and cardiac motion during a beating heart procedure . the use of a robotic assistance can potentially alleviate the need of this level of coordination and provide dexterous manipulation of the interventional tools inside the mri scanner . our group has focused on magnetic resonance imaging- ( mri- ) guided transapical aortic valve replacement [ 2427 ] . in this paper , we report our work on this beating - heart procedure : surgical techniques , medical imaging , medical devices , feasibility of the procedure , and long - term results magnetom espree ( siemens medical solutions , munich , germany ) is used for the intervention . this 1.5-t magnet design , with short ( 120 cm ) and wide ( 70 cm ) bore , gives a clearance of up to 30 cm above the chest of the supine patient and makes surgical access to the patient within the magnet feasible . in addition to providing standard mr sequences , a fully interactive , rtmri system connected to the scanner provides a real - time interactive imaging sequencing . this system comprises an interactive user interface , an operating room large - screen display , gated pulse sequences , and image reconstruction software . multiple oblique slices can be obtained in rapid succession and can be simultaneously displayed in a 3d rendering to provide optimal 3d anatomic information . image contrast , image plane orientations , acquisition speed , 3d rendering , and device tracking can be readily adjusted as needed during scanning . a new self - expanding stent was designed to accommodate conventional stentless aortic bioprostheses ( toronto spv , st . jude medical , minneapolis , mn , or freestyle , medtronic inc . , minneapolis , mn ) ( figure 1 ) . the stent is made of a biocompatible nickel - titanium alloy ( nitinol ) , which assumes a preprogrammed final configuration upon release from the delivery system and exposure to body temperature . the stent has nine rods , three of which are aligned with the valve commissures , and a chevron repeating pattern along the length of the cylinder with flared ends . the fixed length of the stent at both crimped and expanded status prevents stress on the bioprosthetic valve especially at suturing area . the chevron geometry also prevents migration of stent at systole ( high blood pressure ) because of the self - anchoring properties of the chevron spikes . we also implanted balloon - expandable bioprostheses . a stentless bioprosthesis ( toronto spv or freestyle ) was mounted on a commercially available platinum - iridium stent ( cheatham platinum , numed , hopkinton , ny ) ( figure 1 ) . the stented prosthesis was then circumferentially compressed over a balloon - tipped catheter ( numed , 2530 mm od , 50 mm long ) . small austenitic stainless steel fragments ( 0.5 mm ) were welded on the side of both the balloon - expandable and self - expanding stents . this paramagnetic passive marker is visible as a dark signal in the mri and is used to indicate the orientation of the stented prosthesis ( figure 2(a ) ) . a delivery device was developed for holding and delivering the stented prosthesis ( figure 1 ) . the delivery device consists of a straight plastic rod , outside of which is a sheath protecting the stented prosthesis before it is deployed . the diameter of the delivery device is 9.5 mm and fits into a 10 mm trocar . the inner rod has a central channel for a guide wire , balloon catheter , and/or stent retrieving device . this active guide wire is shown as a bright signal in the mri and is also used to indicate the orientation of the stented prosthesis ( figure 2(b ) ) . there is a handle on the inner rod and the sheath , respectively , for the surgeon to hold and manipulate the delivery device . we chose yucatan pigs ( 4557 kgs ) as the animal model for the preclinical studies . the principle reasons for this choice are the similarity to the cardiac anatomy of humans and suitability for long - term studies because growth is somewhat limited compared to domestic strains over the 6 months of followup . after the large animal was intubated and anesthetized , the physician placed the trocar into the apex of the heart . specifically using standard titanium surgical instruments via a 6-cm subxiphoid incision , two concentric purse strings were placed around the apex , through which a 10-mm trocar was inserted into the left ventricle . standard mr sequences were performed to obtain the orientation of the heart , evaluate ventricular and valve function , and locate the native valve annulus and the origin of the coronary arteries . prescanning also allows setting up scan planes to be used for real - time imaging during valve implantation and followup myocardial perfusion and aortic flow imaging . two of these planes were positioned to provide long - axis views of the left ventricle , showing the right coronary artery and left main coronary artery origins , respectively . these digital marks remained visible at all times in the 3d rendering and were used for anatomic reference . the prosthesis was then compressed and placed inside the outer sheath at the distal end of the delivery device . the prosthesis was aligned with the active guide wire in the sheath of the delivery device . the surgeon viewed the real - time imaging on a projection screen while manipulating the deployment device within the animal in the magnet ( figure 3 ) . , the axial slice was shifted as needed to visualize the device and guide proper orientation of commissures with the help of the passive and active markers . the long - axis views were interactively modified to show the path of the delivery device , while keeping the coronary origins in view . both the active wire and the passive marker were used to identify the location and orientation of the prosthesis . the surgeon was in direct contact with the scanner operator by means of headphones and a microphone ( magnacoustics , atlantic beach , ny ) to request changes in the imaging planes as needed . during the procedure , the animals were monitored with an electrocardiogram , oxygen saturation , end - tidal carbon dioxide , systemic and left ventricular blood pressure , and arterial blood gas analysis . in a procedure using the self - expanding prosthesis , the loaded delivery device was first advanced into the ascending aorta . upon release of the stent by retraction of the outer sheath , the chevron - like nitinol cylinder together with bioprosthetic valve expanded to its preprogrammed diameter . retracting and repositioning of the prosthesis were possible before the stent was fully advanced outside of the sheath ( figure 4 ) . in a procedure using the balloon - expandable prosthesis , the balloon is first partially inflated by using normal saline mixed 100 : 1 with an mr contrast agent gd - dtpa ( magne vist , berlex inc . , montville , nj ) ; the position is reconfirmed to be ideal and the balloon is then fully expanded and the prosthesis deployed . after placement of the valve , the trocar was removed and the apex closed with the purse - string sutures . after - placement images were acquired to confirm the positions of the prostheses and the valvular and heart function . phase contrast cine - mri was used to identify flow through the new valve as well as detecting intra- or paravalvular regurgitation . an mr first - pass perfusion scan was performed during intravenous injection of gd - dtpa contrast agent to confirm that myocardial blood flow . the animals were allowed to survive for long - term followup . at 1 and 3 months postoperatively , followup mri scans and transthoracic echocardiography were acquired while at 6 months postoperatively mri scans and confirmatory 2d and 3d transesophageal echocardiography were acquired . retrospectively gated cine mr , phase contrast cine mr , and mr first - pass perfusion scanning during intravenous injection of gd - dtpa contrast agent were repeated at those time points to confirm the position of the prostheses and the valvular and heart function . based on the results seen with a surgeon and human assistant manual approach , we developed an mri compatible robotic surgical assistant system that could more precisely deliver aortic valve prostheses [ 2932 ] . the robotic system consists of an mri compatible robotic arm , a valve delivery module , and user interfaces for the surgeon to plan the procedure and manipulate the robot . the cad sketch of the 9 degree of freedom ( dof ) robotic system which operates in the confined space between the mri bore and the supine patient is shown in figure 4 . an mri compatible innomotion arm ( innomedic , herxheim , germany ) was employed to hold the robotic module and move the valve delivery device on its planned trajectory . the robotic arm has a remote center of motion structure and its configuration fits into a standard closed mri scanner . a robotic module was designed for manipulating a delivery device to position and deploy the prosthesis . the robotic module comprises two linear joints : the translation joint and the insertion joint , as well as a rotational joint . the rotation joint allows the delivery device rotating around its axis to change the orientation of the prosthesis relative to coronary ostia before it is deployed . sole motion of the insertion joint moves only the inner rod of the delivery device , driving the balloon - expandable prosthesis out of the protecting sheath to the desired position . simultaneously retracting the translation joint and advancing the insertion joint at the same velocity keep the inner rod of the delivery device at its location and retracts the protecting sheath back to expose the prosthesis . this simultaneous motion will let the crimped self - expanding prosthesis expand and affix to the desired position . to maintain image quality and prevent local heating in the proximity of the patient , the prototype module was made from nonconductive plastic materials , mr compatible pneumatic actuators ( airpel , norwalk , ct ) , and magnetotranslucent fiber - optical encoders ( innomedic , herxheim , germany ) . the control pc that was placed outside of the mr room communicated with the electronic devices that control pneumatic valves and read encoder signals via the optic network . different interfaces cooperative adjustment , operative plan , and interactive gui adjustments were implemented to suit the needs at the different phases of the procedure ( figure 4 ) . after the physician places the trocar into the subject 's heart , the innomotion robotic arm is then mounted on the mri table and adjusted such that its end effector is close to the trocar port . the robotic module with a fiducial rod attached is mounted on the innomotion arm . the physician uses cooperative hands - on interface to adjust the innomotion arm to insert the fiducial rod into the trocar . once the fiducial rod is in place , the user input sensor is detached and the robot is moved into the bore . in the preoperative phase , the patient undergoes another mri scan for the physician to plan the trajectory of the delivery device . at the same time , another mr sequence is used for system registration . the fiducial rod is then replaced with the delivery device . thus , direct access to the aortic annulus is created . in the intraoperative phase , the physician uses the visual feedback from the rtmri and interactively adjusts and deploys the prosthesis using the robotic module via a gui . a steady - state free precession ( ssfp ) sequence was used with following scanning parameter : tr = 436.4 ms , te = 1.67 ms , echo spacing = 3.2 ms , bandwidth = 1000 hz / pixel , flip angle = 45 , slice thickness = 4.5 mm , fov = 340 283 mm , and matrix = 192 129 . these markers were somewhat difficult to visualize via mri when the stents were fully crimped but became more apparent as the stents were deployed . finally digital markers were placed on the images to identify landmarks and provide surgical references ( e.g. , light blue dots in figure 2(a ) ) . postplacement gated cine mri revealed excellent myocardial function after valve implantation in both long- and short - axis views for animals in whom the valves were appropriately positioned ( figure 5 ) . the phase - contrast cine mr images confirmed good systolic flow with excellent valve leaflet opening and no evidence of turbulence , diastolic regurgitant flow , or paravalvular leak ( figure 5(a ) ) . first - pass perfusion studies demonstrated adequacy of myocardial blood flow after valve placement in all animals following successful deployment . the perfusion results confirmed adequacy of blood flow at the tissue level , indicating proper valve positioning with respect to the coronary ostia ( figure 5(b ) ) . a series of short - term feasibility experiments were conducted in which 42 animals were sacrificed after valve placement and assessment by mri . following the acute studies , 34 animals were enrolled in chronic studies , 11 were implanted with a balloon - expandable prosthesis , and 23 were implanted with a self - expanding prosthesis . total procedure time was 37 and 31 minutes for using balloon - expandable prosthesis and self - expanding prosthesis , respectively . the time from introduction of the prosthesis into the trocar to deployment the stent is fully expanded ( deployment time ) : 74 18 seconds ( mean std . dev . ) and 60 14 seconds ( mean std . dev . ) , respectively . this deployment time was significantly shorter for the self - expanding prosthesis ( p = 0.027 ) . the procedures using balloon - expandable prosthesis take a slightly longer time because of the time used for staging the ballooninflation and the difficulty in orienting the valve knowing that once the balloon was completely inflated there was no margin to allow for adjustment . postmortem pathologic analysis , after sacrifice at 6 months , verified that the implanted prostheses appeared in place in the aortic root . representative radiographs and autopsy confirmation of the self - expanding prosthesis after 6 months implantation are shown in figures 6(a ) and 6(b ) . the average strut fractures for the platinum iridium balloon - expandable stent were 5.0 3.1 ( mean std . dev . ) , while the average fractures for the self - expanding stent were 1.6 2.5 ( mean std . dev . ) the fractures are due to the stent material fatigue and the expansion , contraction , torsion between the aorta and the stent . the mr compatibility of the entire robotic system was evaluated using a 16-cm cylindrical mr phantom inside a 1.5 t siemens espree scanner . the imaging series were taken with ( 1 ) phantom only and ( 2 ) robotic system placed in the magnet and running during imaging . the presence and motion of the robotic system inside the scanner were found to have no noticeable disturbance in the image . the observed snr loss was 8.2% for the entire robotic system placed in the scanner and in motion . we tested the robotic system on a custom - designed phantom for self - expanding prosthesis deployment ( figure 7(a ) ) . the phantom was designed to emulate the dimensions of the valve replacement situation for testing the feasibility of the robotic system . it consisted of a plastic tube with 25-mm diameter , which served as the aorta . this is mounted on one side of a 200 100 100 mm water tank . a spherical joint mounted on a flexible , elastic membrane located on the opposite side of the tank served as the apex . the distance from spherical joint to the end of the plastic tube was 50 mm , which is the typical distance from the heart apex to the aorta annulus as measured in the clinical scenario . the self - expanding prosthesis requires coordinated motion between two coupled pneumatic joints , thus making it a more challenging scenario . we aimed to deploy the self - expanding prosthesis such that its proximal edge is on the edge of the tube under rtmri guidance using robotic system . figure 7(b ) shows the progress of the orientation adjustment and the position adjustment of the prosthesis , as well as the progress of the deployment of the prosthesis . after the prosthesis was deployed , we measured the distance between the edge of the tube and the edge of the prosthesis . the average of absolute system level error over seven trials was 1.14 0.33 mm . despite the requirement of a minithoracotomy , transapical aortic valve implantation is a relatively easy , safe , and straightforward technique . the short and direct access route allows excellent alignment between the prosthesis and the aortic root . with the assistance of the visualization of the active and passive markers on the devices in the mri , the orientation and positioning of the implanted valve are more precise and predictable . real - time mri with proper parameter values provides excellent visualization for intraoperative guidance of aortic valve replacement on the beating heart . it provides better image quality and a complete view of the entire volume of interest more than other competing imaging methods , such as fluoroscopy / angiography , in which some anatomic structures are not visible , and echocardiography , in which the field of view is small and can be obscured by calcification which is frequently the source of the valvular problem . mri - guided surgery also allows direct functional assessments to be made before , during , and immediately after valve implantation that are not obtainable by conventional imaging alone . however , the presence of a strong magnetic field of mri scanner demands all the devices used must be mri safe and compatible . both self - expanding and balloon - expandable prostheses are used in tavr . in our experience , self - expanding stents were easier to position and deploy thus leading to fewer complications during transapical aortic valve replacement . the intrinsic radial force of the self - expanding stent allows for even expansion of the prosthesis . as a result , the self - expanding stent can be retrieved and repositioned before it is fully expanded ; this aids precise placement and diminishes the risk and embolization . the self - expanding stent , with its specific geometric design , handles torsion better , while the balloon - expandable stent has no elasticity and the material is relatively soft leading to more frequent strut fractures . robot assistance can reduce the cognitive load on the physician with improved accuracy and repeatability in transapical valve replacement under mri guidance . the high magnetic field and the confined space of an mr scanner present many technical challenges . the mechatronic components including actuators , sensors , and controllers must be able to work in an accurate , stable , and robust way in an mr environment . materials used for a robotic system should have low magnetic susceptibilities ( comparable with air , water , or human tissue ) , low electrical conductibility , adequate mechanical strength , and good manufacturing properties . the robotic system has been tested on a stable phantom . this phantom is not an ideal replica of the beating heart ; but with proper anatomical dimension between the aortic annulus and the apex , it provides a reasonable situation to validate the coordinated working of the different components of the integrated system before preclinical experimentation . the control strategy and the human machine interface for mri compatible robot systems for medical interventions need to be studied . in the engineering of robots for medical applications , detailed analyses of the functions of the entire system , that is , robot , interfaces and application , taken as single entity , are arguably more important than the individual performance of the subsystems ( robot , surgeon , interfaces , and application , separately ) . thus , having a combination of more than one interface such as ; an image - guided interface , console guided interface , or hands - on interface based on the specific application might yield a higher performance from the entire system . it allows a cohort of patients considered to be at prohibitively high risk for undergoing standard surgical cardiac operation to potentially realize the benefits of a better functioning heart without the morbidity and mortality of a conventional operation . however , minimally invasive cardiac surgical procedures can be technically demanding and more constrained than open procedures . restricted vision , the complexity of instrument manipulation , and difficulty with hand - eye coordination are frequent barriers to the implementation of minimally invasive procedures . we used transapical aortic valve implantation as an example ; demonstrated minimally invasive cardiac surgery can be implemented with the integration of surgical techniques , the technologies of medical images , medical devices , and robotics . the feasibility of the implantation of the transapical aortic valve under real - time interactive mri guidance was successfully demonstrated . the long - term survival experiments further confirm that this minimally invasive surgical technique is safe and robust , ready for translation to a clinical trial . mri provides real - time viewing to allow guidance of procedures in the blood - filled heart without requiring cardiopulmonary bypass and cardiac arrest . real - time noninvasive mr imaging that can provide both anatomic details and functional assessments enables the use of minimally invasive cardiac approaches that may provide patients with a less morbid and more durable solution to structural heart disease . the ability to measure cardiac function online is also an advantage to performing the minimally invasive surgery within the mr scanner . despite its preeminent image quality devices that are used during interventions , such as catheters , are usually not designed to be mr visible or compatible as they often contain ferromagnetic materials or long electrical conductors . the marriage of a medical imaging system and a robot makes the benefit of minimally invasive interventions substantial . an mri compatible robotic assistant system was developed for assisting in transapical aortic valve replacement . different interfaces were implemented to suit the needs at the different phases of tavr procedure . the experimental results show that this robotic system can assist to smoothly deliver the prosthesis under real - time mri guidance with high accuracy . the presence and motion of the robotic system inside the mri scanner were found to have no noticeable disturbance to the image . the performance of using interactive interface to control the robotic system in a beating heart is under further evaluation in an animal study . with the assistance of improvements in engineering technologies such as medical imaging , surgical navigation , and robotic devices , we believe minimally invasive cardiac technique development is a long evolutionary process ; it requires collaborative efforts of physicians and engineers to work cooperatively to fill in the technological gaps .

minimally invasive cardiac surgery is less traumatic and therefore leads to quicker recovery . with the assistance of engineering technologies on devices , imaging , and robotics , in conjunction with surgical technique , minimally invasive cardiac surgery will improve clinical outcomes and expand the cohort of patients that can be treated . we used transapical aortic valve implantation as an example to demonstrate that minimally invasive cardiac surgery can be implemented with the integration of surgical techniques and engineering technologies . feasibility studies and long - term evaluation results prove that transapical aortic valve implantation under mri guidance is feasible and practical . we are investigating an mri compatible robotic surgical system to further assist the surgeon to precisely deliver aortic valve prostheses via a transapical approach . ex vivo experimentation results indicate that a robotic system can also be employed in in vivo models .

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

PMC4980686

akin to developments spurred by the rapid expansion of computer power around 2000 , the burgeoning capacity provided by programmable graphics processing units ( gpus ) has extended the utility of molecular simulations as a practical tool for assessing biophysical processes . notably , gpu - accelerated computing has enabled routine simulations on the microsecond ( s ) time scale , a critical regime on which biological processes including protein recognition , ligand binding , and protein conformational changes occur . access to these longer time scales may reveal flaws in the simulation models that were not previously apparent , driving refinements and leading toward improved predictive power of these models . historically , efforts in force field development have been largely focused on selecting only a subset of the parameters in a complex model for reoptimization , e.g. , reoptimizing certain torsion parameters while keeping the set of atomic charges fixed to improve the accuracy in modeling particular behaviors , while retaining what is already successful . however , such efforts are limited by the accuracy of the unoptimized parameters . however , these refinements may be compensating for deficiencies in the modeling of electrostatic and nonbonded interactions that limit the maximum attainable accuracy of the model . in addition , contemporary force fields have a tendency to borrow from a similar set of values for bond lengths , angles , and atomic radii that were fit many years ago , leading to interdependencies that may be difficult to untangle when optimizing only a portion of the parameters . more recently , semiautomated schemes have been developed to simultaneously optimize hundreds of parameters , thereby enabling the rapid development of new force fields . in particular , the force balance and implicitly polarized charge ( ipolq ) methods have yielded the amber ff15fb and ff14ipq force fields , respectively . these methods rely largely on automated tools for parameter optimization , but still require some amount of manual intervention in the form of fitting set composition or user - specified settings of the fitting algorithm . the engine behind the ipolq workflow is the mdgx module of the amber software package . this module combines its molecular dynamics ( md ) facility with linear algebra routines for solving least - squares problems , manages extensive bookkeeping to organize parameters , provides user control over the fitting process , and interprets statistics to aid in further refinements . the mdgx module contains charge and torsion fitting routines that were built throughout the development of amber ff14ipq , the first complete protein force field based on the ipolq scheme . here , we have developed the new amber ff15ipq force field using the ipolq workflow . the original motivation for the development of ff15ipq was to tackle concerns that its predecessor , ff14ipq , overestimates the stability of salt - bridge interactions a limitation shared with many other contemporary force fields . however , in contrast to recently developed variants of force fields that address such concerns , ff15ipq is far from a limited adjustment of its predecessor . rather , ff15ipq is a complete rederivation , comprising new atomic charges , a greatly expanded torsion parameter set with several new atom types to decouple distinct amino acids , and new backbone angle bending terms . in addition , whereas ff14ipq employed the tip4p - ew model for the solvent in the ipolq scheme , ff15ipq uses spc / eb , a recently developed three - point water model that yields more - accurate rotational diffusion for proteins in solution . the use of a three - point water model instead of a four - point water model , leaving out a virtual site , affords a modest improvement in the speed of cpu - based simulations and a larger acceleration in computing under the amber gpu engine . in addition , the more - accurate rotational diffusion afforded by spc / eb opens new avenues for validating ff15ipq through direct calculation of nmr relaxation parameters . with the aid of gpus and the amber gpu engine , we have extensively validated the force field by running md simulations of peptide and protein systems on the s time scale , yielding over 200 s of aggregate simulation time . we expect ff15ipq , or a close relative , to be valuable for a long time , even as we explore more expensive alternatives by adding virtual sites to both the protein and the standard water model . in addition , we are working to apply the mdgx workflow to other classes of biopolymers such as carbohydrates and nucleic acids , and to small organic molecules . we hope that the sweeping reoptimization made possible by mdgx and tools similar to it will inspire initiatives with other force fields and create complete chemical representations with predictive power in biomolecular simulations . of future interest will be comparisons to contemporary force fields that have been developed in the traditional manner such as amber ff14sb , opls - aa / m , and charmm36 , as well as those developed using alternative sweeping reoptimization schemes , such as amber ff15fb . the implicitly polarized charge ( ipolq ) method is a protocol for parametrizing fixed - charge force fields for solution - phase simulations that is comprised of two main components , implemented in the mdgx program of ambertools . the first component is a protocol for deriving nonpolarizable atomic charges that implicitly represent the energy of polarization by the presence of a solvent such as water . the ipolq charge derivation draws on approximations of dipole interactions in an external electrostatic field to arrive at the optimal nonpolarizable representation of a solute s atomic charges in the presence of a solvent such as water : precisely halfway between the charges that would reproduce the solute s electrostatic field in the gas phase and those that would reproduce the solute s electrostatic field after solvent - induced polarization . this averaging comes about from the fact that the energy of a set of polarizable dipoles in an external field is identical to the energy of a set of fixed dipoles whose polarizations are halfway between the field - polarized dipoles and their gas - phase counterparts . ipolq fits such fixed charges by applying the restrained electrostatic potential ( resp ) method , using a pair of representations of the solute s electrostatic field corresponding to the vacuum and solution phases . while the former representation is straightforward to obtain from qm calculations , the latter is computationally unfeasible using a pure qm representation . instead , the ipolq method represents the polarizing solvent reaction field potential ( srfp ) in its qm calculation using a field of point charges , derived from an md simulation in which water , represented by the model with which the solute will ultimately be simulated , moves in equilibrium around the fixed solute . atomic charges are subsequently fit to reproduce the qm electrostatic potential at a set of grid points surrounding the molecule . as described previously , grid points are selected within the first and second solvation shells , with the inner boundary defined by excluding points for which the energy of the lennard - jones interaction between the solute and a probe representing the water model exceeds a selected maximum cutoff . while , in this work , we have applied equal weights to all of the selected grid points , others have found that more consistent charges may be obtained by applying a weighting function to de - emphasize points close to or distant from the solute . such improvements will be investigated as the ipolq method is applied to other classes of molecules beyond peptides and proteins . the second component of the ipolq method is an extension for the fitting of bonded parameters that accounts for the discrepancy between the desired solution - phase conformational preferences and vacuum - phase qm calculations . this is accomplished by fitting a pair of solute charge sets : one appropriate for the vacuum phase ( qvac ) , and the other for the solution phase ( qsolv ) . in the presence of the qvac charge set , the force field s bonded parameters are fit to reproduce the relative vacuum - phase qm energies of a diverse set of solute conformations . in subsequent simulations in the solution phase , these same bonded parameters are paired with the polarized qsolv charge set with the intention that the difference in the charge sets would account for the difference in solute conformational preferences between the vacuum and solution phases . in contrast to the standard resp method of fitting atomic charges for amber force fields , the ipolq method explicitly considers the influence of the water model on the solute s charge distribution . while the atomic charges of the ff14ipq force field were fit using the tip4p - ew water model , we have elected to fit the charges of ff15ipq using the spc / eb water model . this recently developed water model offers two advantages : in addition to the reduction in computational cost that is obtained by switching from a four - point water model to a three - point water model , this water model has been parametrized to yield accurate rotational diffusion of solvated proteins . a key advantage to performing simulations with accurate rotational diffusion is the ability to directly calculate the nmr relaxation parameters n r1 and r2 and n h heteronuclear noe . these parameters provide information about fast dynamics ( picosecond ( ps ) or nanosecond ( ns ) scale ) of individual backbone n h bond vectors within a protein , potentially offering a powerful means with which to validate md simulations . in principle , these nmr relaxation parameters may be calculated directly from an md trajectory from the autocorrelation functions of the backbone n h vectors . in practice , however , the poor reproduction of protein rotational diffusion in md simulations using popular water models such as tip3p limits the utility of such calculations . this limitation has historically been addressed using approaches such as model - free analysis that attempt to separate the global rotational diffusion of the protein from its residue - specific internal dynamics , comparing only the internal dynamics between experiment and simulation . however , these approaches require extensive fitting of models to the experimental data , and further require that the global and local dynamics occur on separable time scales , limiting their applicability to highly flexible systems such as disordered peptides and proteins . therefore , it would be preferable for md simulations to yield accurate rotational diffusion , such that the simulated and experimental relaxation parameters can be compared directly . our decision to fit the charges of ff15ipq to the spc / eb water model was based on preliminary tests in which we ran a series of 24 simulations of the proteins gb3 , ubiquitin , and binase using two different force fields ( amber ff99sb - ildn and charmm22 * ) paired with four different water models ( tip3p , tip4p - ew , tip4p - d , and spc / eb ) ( see figure s1 in the supporting information ) . consistent with prior published work , tip3p and tip4p - ew yielded rotational diffusion significantly faster than experiment , and spc / eb yielded the most accurate result . earlier versions of mdgx were capable of fitting angle stiffnesses alongside torsions , but recent advances proposed by vanommeslaeghe et al . permit the calculation of both the optimal stiffness constant and equilibrium value from the same linear least - squares problem . the strategy generalizes work by hopkins and roitberg , representing the parabolic angle as the sum of two parabolic basis functions and solving for both scaling coefficients to interpolate the optimal parameters . basis functions were chosen such that their minima lay at 0.2 radians from the equilibria of the original angles , which had been inherited from the amber ff94 force field . as explained by vanommeslaeghe et al . , the optimized angle s stiffness is given by the sum of coefficients solved for each basis function , and its equilibrium is given by the average of the two basis functions minima weighted by their coefficients . restraints on the optimized angle parameters follow from these definitions : a restraint equation setting the sum of the two coefficients ci,1 and ci,2 to a target value ki , such as the stiffness of the original angle in the input force field , will harmonically penalize solutions which depart from the original stiffness value : a similar restraint on the ratio of the two coefficients can be used to penalize solutions that depart from the original equilibrium value ti , if the minima of the basis functions scaled by ci,1 and ci,2 are bi,1 and bi,2 , respectively : as explained in the previous study , these restraint equations will have a more pronounced effect if the dataset contains only 10 data points rather than 1000 data points . therefore , both sides of each restraint equation were scaled by a user - defined constant ( scl ) times the number of instances in which each optimizable angle appeared in the dataset ni , analogous to the scaling constant applied to torsion restraints . the scaling constants may appear to have no effect on the solution to the equations when either these constants are present on both sides of the equation or one side of the equation is zero . however , since the least - squares fit finds an approximate solution to each equation , the scaling constants do , in fact , influence the relative importance of each restraint . in addition , because of the fact that these restraints penalize numerical deviations from the target values but angle stiffnesses and equilibria are expressed in different units by numbers of different scale , a separate scaling factor ( cpl ) was introduced to control the way in which restraints on the equilibria scale , relative to restraints on the stiffness . for example , an cpl of 57 applied to restraints on equilibria would penalize 1 deviations from the original value by the same amount as a 1 kcal/(mol rad ) deviation from the original stiffness constant . after some experimentation , however , we found that , in our very large and heterogeneous datasets , much smaller values of cpl ( 0.51.0 ) result in the best fits to the data while partitioning the changes between equilibria and stiffness constants . alongside the fitting of torsions and angles , several new atom types were added to ff15ipq in order to more accurately capture residue - specific conformational preferences . most protein force fields use gly and ala as templates to develop backbone torsion parameters that are then inherited by other residues . the amber ipolq force fields adopt an unconventional , concerted approach in which , , , , and all other torsions are simultaneously fit to the conformational preferences of all residues in which they appear . while the resulting backbone torsions therefore consider the conformational preferences of residues other than gly and ala , their overall accuracy may decrease as different residues pull the parameters in different directions . within the context of the ipolq fitting method , a set of backbone torsion parameters that more accurately capture the conformational preferences of different residues may be obtained by introducing new atom types , creating decoupled classes of backbone torsions , which are applied to subsets of residues . during the development of ff14ipq , three such classes were introduced : one for gly , one for pro , and one for all other residues . in order to decouple the and torsions of gly , which lacks c and therefore has no and torsions , a unique c atom type was assigned . similarly , the backbone torsions of pro were decoupled by assigning a unique atom type for the backbone n. this additional atom type not only created unique , , and terms for pro , but also a set of separate and torsions for residues preceding pro , thereby enabling the force field to capture the unique conformational preferences of these contexts . the remaining residues were further divided into three subclasses based on their c types , which determine the applied and torsions . the c types of ff14ipq yielded four subclasses : ( i ) flexible positively charged residues ( arg , lys ) , ( ii ) residues whose c atoms are bonded to two heavy atoms and whose side - chains are not aromatic ( asn , asp , cys , gln , glu , leu , met , ser ) , ( iii ) residues whose c atoms are bonded to three heavy atoms ( ile , thr , val ) , and ( iv ) all other residues ( ala , his , phe , trp , tyr ) . notably , this last subclass yielded and torsions shared between ala and the bulky aromatic residues . this unusual coupling was a consequence of the force field s lineage from ff12sb , where refitting of x1 torsions alongside fixed , , , and did not require a unique c type for the aromatic residues , which already have unique c types that yield unique x1 . in order to further improve the accuracy of residue - specific conformational preferences in ff15ipq , several new atom types were added to further decouple the backbone torsion parameters of different residues , leading to a total of five backbone classes . in order to restrict each class of backbone torsions to a single set of scaled 14 electrostatic terms , negatively charged ( asp , glu ) and positively charged ( arg , lys ) residues have been given unique c types , decoupling their , , , and from those of the neutral residues . while the backbone n of pro was decoupled in ff14ipq , it retained a shared torsion ; to break this dependency , pro has now been assigned a new c atom type . finally , the coupling between ala and the bulky aromatic residues has been removed by assigning his , phe , trp , and tyr a unique c type , decoupling their and terms from ala . this decoupling divides the neutral residues into four subclasses : ( i ) ala , ( ii ) residues whose c atoms are bonded to two heavy atoms ( asn , cys , gln , leu , met , ser ) , ( iii ) residues whose c atoms are bonded to three heavy atoms ( ile , thr , val ) , and ( iv ) bulky aromatic residues ( his , phe , trp , tyr ) . the backbone torsion classes of the 28 residue forms supported by ff15ipq are listed in table s1 in the supporting information . to compare the accuracy of ff15ipq in modeling the stability of protein salt bridges to its predecessor ff14ipq and contemporary force fields , we simulated the association of three pairs of oppositely charged amino acid side - chain analogues : guanidinium cation / acetate anion ( arg / asp ) , butylammonium cation / acetate anion ( lys / asp ) , and imidazolium cation / acetate anion ( his(+)/asp ) . for comparison , such simulations were carried out using the polarizable force fields charmm drude-2013 and amoeba , in addition to other fixed - charge force fields that we had previously tested using these three model systems . simulations with ff15ipq , ff14ipq , and amoeba were carried out using the amber 15 software package , while those with charmm drude-2013 were run with namd 2.10.0 , following a protocol analogous to that used for the previously evaluated fixed - charge force fields ( full details are provided in the supporting information ) . systems were constructed to be consistent with the experimental conditions under which the association constants ( ka ) of guanidinium acetate and butylammonium acetate have been measured , i.e. , each system consisted of 100 molecules of cation ( guanidinium , butylammonium , or imidazolium ) , 2 molecules of acetate , and 98 chloride counterions solvated by 18 000 water molecules . for the fixed - charge force fields , parameters of the side - chain analogues were based on those of the complete amino acids . for the charmm drude-2013 polarizable force field , parameters of guanidinium , imidazolium , and acetate were those distributed alongside the force field . since methylammonium rather than butylammonium was used as the analogue of lys during the development of drude-2013 , the butylammonium acetate system was not tested with this force field . for the amoeba force field , parameters of guanidinium , imidazolium , and acetate were generated using the poltype derivation protocol ( details are provided in the supporting information ) . as it was with charmm drude-2013 , the butylammonium acetate system was not tested with amoeba . for all of the simulations mentioned above , the probability that an acetate molecule was bound to one or more cation molecules was calculated by assigning each pair to either the bound or the unbound state . for each force field and pair of side - chain analogues , definitions of the unbound and bound states were based on the potential of mean force ( pmf ) as a function of the minimum distance between nitrogen atom(s ) of the cation and the oxygen atoms of acetate . in particular , the cutoff between the bound and unbound states was defined as the point of inflection between the free energy minimum of the bound state ( 2.53 ) and the free - energy maximum , which corresponds to the desolvation barrier ( 33.5 ) . pairs whose minimum n o distances were below this cutoff were assigned to the bound state , while those beyond were assigned to the unbound state . in addition to species in which a single acetate molecule was bound to a single cation molecule , forming a 1:1 complex ( e.g. , the guanidinium / acetate complex ) , species in which acetate was bound to two or more cation molecules ( e.g. , the 2:1 diguanidinium / acetate complex ) were observed and counted separately . the atomic charges of ff15ipq were fit using the ipolq module of mdgx , as described previously for ff14ipq . during charge fitting , each amino acid was represented by a blocked dipeptide including acetyl ( ace ) and n - methylamide ( nme ) caps ; terminal forms were represented by omitting one of the blocking groups , while the disulfide form of cysteine ( cyx ) was represented by a pair of dipeptides linked by a disulfide bond . to expand on the set of amino acids and protonation states that were supported by ff15ipq , atomic charges were also derived for the following : the n- and c - terminal forms of protonated aspartate ( ash ) and glutamate ( glh ) , the c - terminal form of neutral lysine ( lyn ) , the terminal and nonterminal forms of deprotonated cysteine ( cym ) , and the noncanonical amino acid norleucine ( nle ) . each solute of interest was solvated in a cubic box of spc / eb water with a clearance of 10 between the solute and the edge of the box , and subjected to a high - temperature md simulation at 450 k , from which were collected a set of 20 conformations . each conformation was subsequently re - equilibrated at 298 k before being input to the ipolq module of mdgx . this module was used to run an md simulation with the solute fixed , during which the coordinates of surrounding solvent molecules were collected and used to generate a collection of point charges representing the solvent reaction field potential . this collection consists of an inner cloud of point charges taken directly from the coordinates of solvent molecules within 5 of the solute , and three outer shells of point charges fit to reproduce contributions to the solvent reaction field potential from the infinite periodic system beyond 5 . a pair of qm calculations for the solute were then run at the mp2/cc - pvtz level of theory : one in vacuum and the other including the solvent reaction field potential . as modeled by the collection of point charges . these calculations were run using the orca 3.0.3 software package for each conformation of each residue , requiring over 3000 density calculations . the resulting densities were then input to mdgx s fitq module , yielding a pair of charge sets : one valid for simulation under vacuum ( qvac ) and the other for simulation in solution ( qsolv ) . the bonded parameters of ff15ipq were fit to reproduce the relative vacuum - phase qm mp2/cc - pvtz potential energies of a set of diverse conformations of short peptides using an iterative cycle of refinement , similar to that used for its predecessor , ff14ipq . this cycle involved the following steps : ( i ) md simulations were carried out to generate a set of peptide conformations , ( ii ) these conformations were subjected to energy minimization in vacuum using the molecular mechanics ( mm ) energy function with qvac and the current generation of bonded parameters , ( iii ) qm energies of the energy - minimized conformations were calculated , ( iv ) the conformations and energies were used to fit an improved set of bonded parameters , and ( v ) steps ( i ) through ( iv ) were repeated to fit the next generation of bonded parameters . in this way , subsequent generations of the force field learned from the biases of their ancestors , provided those biases were captured in the qm energies of the additional conformations that resulted from step ( i ) of the iterative cycle . during the development of ff14ipq , selected conformations from an initial fitting set of 28 000 were subjected to energy minimization with each new generation of bonded parameters to yield new conformations , accumulating a total of 65 000 structures and single - point energies . the first generation of ff15ipq fitting data was created by pairing ff14ipq with generalized born implicit solvent md simulations of amino - acid dipeptides at 450 k , followed by vacuum energy minimization of many snapshots from each simulation . while we have not tested how well ff14ipq behaves with implicit solvent , the purpose was to capture any spurious conformational preferences that might remain in the original force field . in addition , we included 1400 conformations of the ace - ala - pro - ala - nme tetrapeptide , while the second generation added numerous tripeptides containing gly , and conformations of the disulfide - bridged cyscys system ( among the largest of all the systems used in qm single - point - energy calculations ) . the next three generations of refinement were designed to cover sampling of the multiple classes of backbone parameters applied to different residues , as described in section 2.4 . in order to ensure sampling of diverse backbone conformations , conformations were generated by progressively restraining and at 20 intervals , using a 16 kcal / molrad harmonic restraint over the course of the md simulation , yielding 324 conformations of each . since the unique backbone nitrogen type of pro creates unique and terms for preceding residues , the third generation of conformations consisted of 51 pro - containing tripeptides for which the non - pro residue s and were restrained . at this point in development , it was decided to branch the positively- and negatively charged residues into unique backbone classes , and as such the fourth and fifth generations of refinement consisted of 57 tripeptides containing the charged residues asp , glu , cym ( deprotonated cys ) , arg , lys , and hip ( doubly protonated his ) , in which and of the charged residues were restrained . in order to cover the unique backbone parameters applied to the terminal forms of each residue , additional conformations were added for a set of 78 terminal nxaa - nme and ace - cxaa monopeptides . for these terminal systems , scans of either the unique of the n - terminal forms or the unique of the c - terminal forms since the unique backbone nitrogen types of the n - termini and pro in tandem yield an additional set of terms for nxaa - pro , scans of were run for an additional set of nxaa - pro - nme dipeptides . finally , in order to cover the unique backbone and terms of the amide blocking group ( nhe ) , scans of and were run for 17 ace - xaa - nhe dipeptides , yielding 324 conformations of each . during these three generations of refinement , 60 000 conformations were added to the ff15ipq fitting set . after the fifth generation of refinement , support for the fitting of angle equilibria and force constants alongside torsions was implemented in mdgx , and subsequent generations emphasized comprehensive sampling of backbone angles . the sixth , seventh , and eighth generations of refinement consisted of perturbations of the angles around n , c , and c. starting from an initial conformation , a selected angle of interest was subjected to a random perturbation within a range of 20 of its original equilibrium value ( as inherited from the ff94 force field and retained in contemporaries such as ff14sb ) . target values for the other angles around the same central atom were then chosen by taking their initial values and adjusting them such that the total sum of angles around the central atom was appropriate for the known geometry ; target sums of 360 for planar geometry around n and c and 660 for tetrahedral geometry around c were used . during subsequent mm minimization , the target values for these angles were restrained using 256 kcal / mol rad harmonic restraints . during the eighth and final generation of refinement , angle perturbations were resampled in the context of new scans of and backbone torsions at 10 intervals for each ace - xaa - nme dipeptide , yielding 1296 conformations of each , alongside additional sampling of terminal monopeptides . during these three generations , 125 000 conformations were added , yielding a final fitting set of ff15ipq consisting of > 250 000 single - point qm energies , which is over four times larger than that used for ff14ipq . as done previously for ff14ipq , the torsion parameters of ff15ipq were fit using a linear least - squares fit implemented in the param module of mdgx ; extensions to the module for angle fitting are described in section 2.2 . this module selects a set of torsional barrier heights , angle equilibria , and angle stiffnesses that best reproduce the relative conformational energies of the systems included in the fitting set . during the fitting process , the fourier series lengths and phase angles of the torsional terms were not optimized , and phase angles were set to either 0 or 180 to enable the development of parameters that are transferable to alternative chiralities . all backbone , , , , and side - chain x torsions of nonterminal forms of the amino acid residues were allocated four terms in their fourier series . torsions unique to the terminal forms of residues and residues preceding pro were restricted to only three terms since these terms were less exhaustively sampled in the fitting set . this restriction was applied to limit the risk of overfitting . while all torsion parameters of residues in the fitting set were fit , only angles in which the central atom was the n , c n type of pro introduces a large number of parameters that are dependent on the preceding residue . in order to avoid overfitting torsional barrier heights , torsions were restrained toward 0 with a force constant of 2 10 kcal / mol . similarly , angles were restrained to their original values , inherited from ff94 , with the equilibria and stiffness force constants set to 5 10 kcal / mol and 2 10 kcal / mol , respectively . to characterize the backbone conformational preferences of ff15ipq in explicit spc / eb water , we carried out umbrella sampling simulations of blocked tetrapeptides ace - ala - xaa - ala - nme , calculating the potential of mean force as a function of the backbone and torsions of the central residue xaa . in order to identify differences in the conformational preferences between ff15ipq , its predecessor ff14ipq , and contemporary force fields , simulations of ace - ala - ala - ala - nme were carried out using the amber force fields ff15ipq , ff14ipq , and ff14sb ; the opls force field opls - aa / m ; and the charmm force fields charmm36 and drude-2013 . analogous simulations were carried out to compare the conformational preferences of other central amino acid residues using the amber ff15ipq , ff14ipq , ff14sb , and charmm36 force fields . the backbone and torsions of the central residue were restrained in a series of 1296 windows spaced at 10 intervals , using a harmonic penalty function with a force constant of 8 kcal / mol rad . each window was seeded from a continuous , incrementally restrained simulation , and sampled for 2.0 ns , following a 0.2 ns equilibration . from each set of 1296 windows were reconstructed the unbiased potentials of mean force using the weighted histogram analysis method ( wham ) . to validate ff15ipq as a general force field for peptides and proteins , extensive md simulations on the s time scale were carried out for a variety of benchmark systems consisting of both structured and disordered peptides and proteins . for each system , the amino - acid sequence or pdb code , sources of initial coordinates , and temperatures maintained throughout the simulations are listed in table 1 . hp35 double - norleucine mutant mutant ( lys24nle , asn27his , and lys29nle ) . the p53 peptide used contained residues 1729 of the full - length protein and included an n - terminal acetyl ( ace ) and c - terminal amide ( nhe ) blocking group . mdm2 included residues 25109 of the full - length protein , omitting a mobile n - terminal region unresolved in the crystal structure . the n- and c - termini of mdm2 were blocked with acetyl ( ace ) and n - methylamide ( nme ) blocking groups , respectively . in order to accurately match the amino acid sequences used in nmr experiments , residues not resolved in the crystal structure were built using avogadro . residues stsaa were appended to the c - terminus of the s - peptide , and sss to the n - terminus of the s - protein . in addition , ga residues were appended to the n - terminus of the s - peptide , representing a cloning artifact present in the nmr experiments . these residues were not restrained during equilibration of the system . given that the nmr experiments on the s - peptide / s - protein complex were conducted at ph 3.7 , which is close to the average pka values of asp ( 3.7 ) and glu ( 4.1 ) , we elected to run our simulations with negatively charged asp and neutral glu . as done for ff14ipq , we validated ff15ipq by simulating penta - alanine ( ala5 ) , the -helical k19 peptide , the gb1 -hairpin from the c - terminal fragment of protein g , the designed -hairpin chignolin , trp - cage , gb3 , and lysozyme . we also carried out simulations of the -helical ( aaqaa)3 peptide , the cln025 mutant of the chignolin -hairpin , the double - norleucine variant of the villin headpiece subdomain , bovine pancreatic trypsin inhibitor ( bpti ) , ubiquitin , and binase . in order to evaluate the ability of ff15ipq to model disordered proteins , we simulated two classic systems for studying the binding processes of disordered peptides that fold only upon binding their partner proteins : ( a ) the n - terminal p53 peptide and mdm2 oncoprotein , and ( b ) the s - peptide and s - protein cleavage products of the rnase a protein . both of these peptides fold into -helical conformations only upon binding their partner proteins . simulations were performed with these peptides both in isolation and in complex with their protein binding partners . simulations of benchmark systems were carried out using the gpu implementation of the pmemd module in the amber 15 software package . each system was solvated in a truncated octahedral box of spc / eb explicit water with a 12 buffer for the disordered peptide / protein systems and 10 buffer for all other systems . prior to production simulation , each system was subjected to energy minimization , followed by a three - stage equilibration . in the first stage , a 20 ps simulation of the energy - minimized system was carried out at constant temperature while restraining the solute heavy atoms to their initial positions using a harmonic potential with a force constant of 1 kcal / mol . in the second stage , a 1 ns simulation was carried out at constant pressure with the same harmonic position restraints . finally , an additional 1 ns unrestrained simulation was carried out at constant temperature and pressure . temperatures were maintained at selected values ( between 270 k and 370 k ) , using a langevin thermostat ( frictional constant of 1 ps ) , while pressure was maintained at 1 atm using a monte carlo barostat ( 200 fs between attempts to change the system volume ) . van der waals and short - range electrostatic interactions were truncated at 10 ; long - range electrostatic interactions were calculated using the particle mesh ewald method . to enable at least a 2 fs time step , bonds to hydrogen were constrained to their equilibrium values using the shake and settle algorithms . for the k19 , ( aaqaa)3 , gb1 hairpin , chignolin , and cln025 systems , hydrogen mass repartitioning was used to enable the use of longer timesteps . in particular , the masses of solute hydrogen atoms were increased by a factor of 3 , and that of their attached heavy atoms decreased by a corresponding amount such that the total mass remained constant ; the masses of water molecules were not repartitioned . this mass repartitioning scheme enables a 4 fs time step for simulations at 300 k ; for simulations at > 300 k , shorter timesteps were used and set to be equal to 1200 k fs , divided by the set temperature . diagnostics included dssp , rotational diffusion , and nmr relaxation calculated by the ired method . to evaluate the accuracy of ff15ipq in modeling the strengths of protein salt bridges , we simulated the association of three pairs of oppositely charged amino acid side - chain analogues : guanidinium cation / acetate anion ( arg / asp ) , butylammonium cation / acetate anion ( lys / asp ) , and imidazolium cation / acetate anion ( his(+)/asp ) . for each salt bridge , the resulting probability of an anion binding to one or more cation molecules ( pbound ) was compared to experiment ( if available ) , as well as those of six other fixed - charge force fields , including ff14ipq , ff14sb , ff03 , charmm22 * , charmm36 , and opls_2005 , and two polarizable force fields ( charmm drude-2013 and amoeba ) . the original motivation for the development of ff15ipq was to correct for the overstabilization of protein salt bridges by its predecessor , ff14ipq . during the development of ff14ipq , the lennard - jones radii of several polar heavy atoms were refit to reproduce the experimental solvation free energies of side - chain analogues . although the resulting set of radii were initially intended to be applied globally , several of the larger radii resulted in increased 14 repulsion during torsion fitting , which made the torsion parameters more difficult to fit . to overcome this difficulty , mixed lennard - jones combining rules ( called ljedit within amber software or nbfix within charmm ) were applied to these polar groups , assigning different radii for their solute solvent interactions from those used for their solute solute interactions . for example , for the carboxylate oxygen atoms of the side - chains of asp and glu larger lennard - jones radii for interactions with water were used than for interactions with solute atoms . an undesirable effect of this strategy , however , was the overstabilization of salt bridges to the point that in our simulations each acetate molecule was bound to three or more cation molecules ( e.g. , guanidinium ) for most of the simulation ( see figure 1 , as well as figure s2 in the supporting information ) . essentially , the larger radii used for solute solvent interactions forced the carboxylate group out of solution and into interactions with available solute atoms . probability of binding ( pbound ) between acetate and one or more molecules of three cationic side - chain analogues using seven fixed - charge and two polarizable biomolecular force fields , each paired with either the water model with which it was derived or that with which it is most - commonly used . the pbound values corresponding to the experimentally determined ka values of guanidinium acetate and butylammonium acetate are depicted as horizontal gray bars ; no experimental value is available for the imidazolium acetate system . results for the charmm36 , charmm22 * , opls_2005 , amber ff14sb , and amber ff03 force fields are taken from previous simulation studies . for ff15ipq , we addressed the problem of overstabilized salt bridges by discarding the mixed lennard - jones radii of ff14ipq and instead applied empirical corrections to the radii of polar hydrogen atoms bonded to nitrogen ( atom type h ) in both the protein backbone and side - chains ( note that the original value of 1.07 for this atom type may equivalently be expressed as an r * of 0.6000 , and the details of its fitting appear to have been lost to history ) . these corrections were determined from simulations of the three oppositely charged side - chain analogue systems with h ranging from the ff94 value of 1.07 up to 1.5 , calculating the probability of salt bridge formation ( pbound ) , and comparing this probability to that from experiments . based on the results ( figure s2 ) , we selected a value of 1.3 for nitrogen - attached hydrogens in both the protein backbone and side - chains . for guanidinium acetate , we found that a further increase in to 1.5 for the side chain of arg was necessary to achieve satisfactory agreement with the experimental value of this system . as shown in figure 1 , ff15ipq yields pbound values that are among the most reasonable , relative to the other fixed - charge force fields that were tested ( ff03 , ff14sb , ff14ipq , opls_2005 , charmm22 * , and charmm36 ) . for guanidinium acetate , our results with ff15ipq are roughly consistent with charmm22 * and ff03 , which we had previously found to provide the most accurate modeling of this system ( note that the corresponding figure of our previous work omitted complexes including multiple cation molecules bound to a single anion molecule and therefore under - represented the extent to which the force fields overstabilize salt bridges ) . for butylammonium acetate , ff15ipq yields a pbound value that is slightly higher than that of amber ff03 , but similar to those of charmm22 * and ff14sb . for imidazolium acetate , ff15ipq yields a pbound that is higher than those of charmm22 * and ff03 , but similar to that of ff14sb . of particular note is that charmm22 * was also parametrized to reproduce the experimental association of guanidinium acetate , but via adjustments to the atomic charges of only the side - chains of arg , asp , and glu ; as a result , these adjusted parameters are inconsistent with the rest of the force field , whose charges had been fit years earlier , using a different method . along the same lines , a recently developed variant of the amber ff99sb - ildn force field has involved the application of mixed lennard - jones combining rules exclusively to interactions between the side chains of arg , asp , and glu . in contrast to the posthoc adjustments of these two other force fields , our approach involves first adjusting the lennard - jones radii , followed by refitting of atomic charges and bonded parameters . this approach which has been an onerous one in the past has been significantly streamlined by the mdgx software . we note that all of the fixed - charge force fields are outperformed by the more expensive , polarizable charmm drude-2013 and amoeba force fields . while it is likely that much of their superior performance results from the more complex charge model for the solutes , it is also possible that the solute solvent interactions which compete with solute solute interactions are more accurately represented by the use of polarizable water models . in particular , fixed - charge water models similar to the spc / eb model used here have recently been found to generally underestimate the strength of solute solvent interactions , and it is possible that this limitation of the water models restricts the accuracy with which the solute models may represent salt bridges . a key metric for assessing the accuracy of the torsion and angle parameters of ff15ipq was the ability to reproduce the target qm potential energy surface . figure 2 shows the distribution and root - mean - square error ( rmse ) of ff15ipq energies , with respect to their target qm potential energies for the 20 canonical amino acids . the rmse values for all neutral residues are < 1.3 kcal / mol , while those of the negatively charged residues asp and glu are < 1.9 kcal / mol , and those of the positively charged residues arg and lys are < 2.4 kcal / mol . as shown in the supporting information ( figure s3 ) , the neutral forms asp , gln , and lys ( ash , glh , and lyn , respectively ) have rmse values that are consistent with the other neutral residues , suggesting that the increased rmse values of lys and arg , relative to uncharged residues , are related to their net charge , rather than to their additional flexible x torsions . optimization of the backbone angle parameters introduced a 5%15% improvement in rmse and enabled expansion of the fitting set by more than 4-fold without sacrificing the ability to reproduce those parts of the qm potential energy surface represented in the original dataset ( see the section entitled timeline of development in the supporting information ) . distributions of residuals of relative molecular mechanical energies , with respect to their qm target potential energies , for 18 ace - xaa - nme dipeptides and the ace - ala - ala - ala - nme and ace - gly - gly - gly - nme tetrapeptides . the 25th , 50th , and 75th percentiles are represented by horizontal lines , and root - mean - square values are represented by white circles . each dataset is colored based on its corresponding backbone torsion class . to assess the accuracy of ff15ipq in modeling the backbone conformational preferences of proteins within computationally tractable systems , we carried out a series of simulations of short peptides that may be affordably simulated to convergence . initially , we focused on simulations of the ace - ala - ala - ala - nme tetrapeptide , for which we calculated the pmf as a function of the backbone and torsions of the central residue . figure 3 shows the results for ff15ipq , its predecessor ff14ipq , and several contemporary force fields . relative to ff14ipq , ff15ipq has larger free energy barriers ( by 1 kcal mol ) between the well ( 70 , 20 ) and well ( 80 , 60 ) and between the well ( 150 , 150 ) and ppii well ( 70 , 140 ) . in addition , ff15ipq has a more clearly defined well ( 140 , 50 ) . on the left half of the ramachandran plot , 40 ) has decreased slightly , and that of the well ( 70 , 40 ) has decreased by 1 kcal / mol , while the pii well ( 60 , relative to the ff14sb and charmm36 force fields , ff15ipq shows similar and ppii well depths , although ff14sb and charmm36 do not exhibit or wells and the precise positions of the various wells differ between the force fields . larger differences are observed relative to the opls - aa / m and polarizable charmm drude-2013 force fields , which have shallower and deeper wells , respectively . potentials of mean force for the central residue of blocked alanine tetrapeptides , as a function of backbone and torsions of the central residue using five fixed - charge force fields and one polarizable force field . each force field was paired with either the water model with which it was derived or that with which it is most commonly used . next , we extended our validation of ff15ipq by examining residue - specific backbone conformational preferences . in particular , we carried out simulations of ace - ala - xaa - ala - nme tetrapeptides containing each of the 20 canonical residues at the central position , including the 25 protonation states of these residues that are supported by the force field . for comparison , analogous simulations were carried out using the ff14ipq , ff14sb , and charmm36 force fields . the resulting / backbone torsional preferences of the central residues were then compared to those of ala - xaa - ala obtained from the neighbor - dependent ramachandran distribution ( ndrd ) dataset , derived from conformations observed in the loop regions of proteins ( non--helix/-sheet secondary structures ) . the ndrd dataset is drawn from a collection of 3000 high - resolution crystal structures in the protein data bank , and accounts for the influence of preceding and following residues on the / backbone torsional preferences of the central residue . given the considerable differences in the contexts of our simulations and the ndrd experimental data set , i.e. , solution vs crystal environment , we focused solely on qualitative differences between the simulated and experimental conformational preferences of each peptide . in particular , we compared the conformational preferences of peptides containing a nonalanine central residue , relative to that of the reference ace - ala - ala - ala - nme peptide . generally , both ff15ipq and ff14ipq show greater variation between amino acids than ff14sb and charmm36 , which apply the same backbone torsions to all residues ( figure s4 in the supporting information ) . several differences between ff15ipq and ff14ipq are apparent . for the neutral residues whose c atoms are bound to two heavy atoms , the clearest difference is the decreased favorability of the 180 < < 90 region for asn , gln , leu , and met ; ff15ipq is more consistent with ndrd distributions in which such conformations are rare , because of the broader sampling of such uncommon backbone conformations in the ff15ipq fitting set . an exception is ser , which retains this region and exhibits overall broader sampling , in contrast to the ndrd distribution , in which conformations are restricted largely to the canonical wells . for the neutral residues whose c atoms , the ndrd dataset shows increased conformational preferences in the region and in the region adjacent to the well , centered at 120 , 60. these preferences are captured by both ff14ipq and ff15ipq , but the lower region is erroneously disfavored by both ff14sb and charmm36 . while ff15ipq is improved relative to ff14ipq by disfavoring the l well of thr , the near - absence of sampling of this well in the ndrd distribution suggests that it may still be too favorable , relative to the experiment . differences between ff15ipq and ff14ipq for the bulky aromatic residues are less pronounced ; the conformational preferences of these residues may be more dependent on sterics as modeled by the lennard - jones parameters , which have not been changed in ff15ipq from those of ff14ipq . the greatest differences between ff15ipq and ff14ipq are observed for the negatively charged residues asp and glu , which have been granted their own , , , and torsions in ff15ipq . these residues largely restrict sampling to the of ppii , , and wells , lacking clearly defined wells and any wells on the right side of the ramachandran plot . the differences for the positively charged residues arg and lys are much smaller , likely because these residues were already assigned unique and torsions in ff14ipq . to complement the above qualitative comparisons , we obtained quantitative measures of the accuracy of ff15ipq s backbone conformational preferences by calculating j - coupling constants for the ala5 peptide and comparing these values to experiment . this peptide was the focus of a study by best et al . in which multiple force fields were compared , in terms of their ability to reproduce experimental j - coupling constants using the karplus equation and three different sets of karplus coefficients : the original coefficients , as used by graf et al . , and two sets of dft - based coefficients ( dft-1 and dft-2 ) by case et al . in this study , a suggested criterion for a high - quality force field is that the value between calculated and experimental j - coupling constants should be 2.25 for all three sets of karplus coefficients . three useful points of reference are the recently developed ff14sb , charmm36 , and ff03w force fields , which were empirically corrected to improve reproduction of experimental ala5 j - coupling constants . the ff14sb force field yielded values of 0.9 and 1.2 with the original and dft-2 coefficients , respectively , but a higher of 2.7 with the dft-1 coefficients ; charmm36 and ff03w were tested only with the dft-2 coefficients , yielding values of 1.16 and 0.9 , respectively . as shown in table 2 , ff15ipq yields values of = 0.53 with the original coefficients , = 1.08 with the dft-2 coefficients , and = 0.67 with an additional set of karplus coefficients from lindorff - larsen et al . however , similar to ff14sb , we obtained a higher value of 2.91 with the dft-1 coefficients ; in our case , the higher value is driven primarily by a single outlier that deviates greatly from the experiment , jhnc. based on results from preliminary versions of ff15ipq ( see the timeline of development section in the supporting information ) , it appears that lower values with the original karplus coefficients may come at the expense of higher values with the dft-1 coefficients . notably , ff15ipq which employs a general parametrization to reproduce qm potential energies performs at least as well as ff14sb , charmm36 , and ff03w , which have been parametrized specifically to reproduce ala5 j - coupling constants . all four force fields yield results that are much improved , relative to force fields developed only a few years ago . uncertainties on values represent one standard error of the mean , calculated using a block averaging method . uncertainties on individual j - coupling constants are omitted for the sake of clarity . note that the calculated j - couplings are dependent on the ratios of various backbone conformations , between which transitions may be relatively rare . in our studies of ala5 , we found that 1.5 s of aggregate simulation time , which we had previously used in our validation of ff14ipq , did not yield sufficiently precise calculations of the j - couplings . although the j - couplings may appear to be converged based on their relatively small statistical variances ( evaluated using block averaging ) , these variances may be misleading . for example , we observed what appeared to be small , but statistically significant differences in the j - couplings between simulations run with and without hydrogen mass repartitioning after 1.5 s of simulation , but these differences ultimately disappeared after 6 s ( see the timeline of development section in the supporting information ) . the extensive sampling needed to obtain converged j - couplings illustrates the challenge of mapping the conformations of just a few residues using brute - force md simulation . to assess the propensity of ff15ipq to form -helices , we studied the temperature - dependent behavior of two model -helical peptides : k19 and ( aaqaa)3 . both peptides are variants of the motif ( ala - ala - xaa - ala - ala)n , in which xaa is lys in k19 and gln in ( aaqaa)3 ; their sequences are listed in table 1 . for each peptide , we carried out six 4-s simulations at different temperatures and monitored the formation of various types of secondary structure . as shown in figure 4 , both peptides undergo multiple folding and unfolding events , although our simulations are not sufficiently long to obtain converged estimates of the probability of adopting -helical conformations . qualitatively , k19 adopts -helical conformations for a greater proportion of the simulation than ( aaqaa)3 , which is consistent with the experimental observation that k19 and ( aaqaa)3 are 40% and 20% -helical , respectively , at 300 k. both peptides transiently form -sheet contacts , which do not appear to be stable for more than 100 ns , indicating that ff15ipq correctly identifies the favored secondary structures of these peptides . secondary structure of model -helical peptides ( a ) k19 and ( b ) ( aaqaa)3 at various simulated temperatures over the course of 4-s simulations . while the two peptides differ in length , the observed difference in -helical stability is likely due to parameters of gln and lys residues at the central positions of the peptides . in our umbrella sampling simulations of tetrapeptides , which are too small to form an -helix ( figure s4 ) , we observed a broader , deeper well for lys than for gln , suggesting that the observed difference in -helical stability between the two peptides has already been built - in to ff15ipq at the residue level . in addition , the two residues have different backbone charges : gln shares its n , h , c , and o charges with the other neutral residues , and lys shares its charges with the positively charged residues . while the backbone h charges for neutral and positively charged residues are similar , the backbone o of the positively charged residues is 0.05 e more negative than that of the neutral residues , which may result in more stable hydrogen bonding . in order to assess the stability of -sheet structures in ff15ipq , we simulated three model -hairpin systems : the gb1 hairpin , the designed peptide chignolin , and its hyper - stable variant cln025 . we simulated the gb1 hairpin at 6 temperatures , ranging from 275 to 325 k , and we simulated cln025 at 10 temperatures , ranging from 280 k to 370 k ; we simulated chignolin only at 298 k. figure 5 shows the secondary structures observed during 4-s simulations of these systems . as with our simulations of the -helical peptides , our -hairpin simulations are not sufficiently long to precisely quantify secondary structure stability , although qualitative trends may be identified . as shown in figure 5a , the gb1 hairpin is metastable over the tested temperature range of 275325 k , and in two of our simulations unfolds and refolds . our simulations at 285 k are in qualitative agreement with the experiment , which have indicated that the gb1 hairpin is 85% folded at 275 k , 50% folded at 295 k , and 20% folded at 325 k. however , an anomaly is observed in our 275 k simulation , in which the gb1 hairpin unfolds after 200 ns and does not refold . this unfolding event may simply be an artifact of our limited sampling that would disappear if the simulations were to run to convergence . alternatively , it may reflect limitations of the spc / eb water model at temperatures distant from those at which it was parametrized ; while the temperature - dependent behavior of spc / eb has not been characterized , to our knowledge , three - point water models including the parent spc / e water model are known to poorly reproduce the temperature dependence of properties such as density . secondary structures of model -hairpin peptides ( ( a ) gb1 hairpin , ( b ) chignolin , and ( c ) cln025 ) at various simulated temperatures over the course of 4-s simulations . in contrast , our simulations of chignolin and cln025 suggest that these -hairpin systems may be more stable than observed experimentally . as shown in figure 5b , chignolin maintains its -hairpin configuration throughout our 4-s simulation at 298 k , including two hydrogen bonds in an antiparallel sheet configuration , while , experimentally , the peptide is only 60% folded at this temperature . chignolin s hyper - stable variant cln025 has an experimental melting temperature of 343 k. as shown in figure 5c , in our simulations at temperatures ranging from 280 k to 370 k , we observe unfolding and refolding events at several temperatures , although the overall folded population is larger than that measured experimentally . in particular , cln025 is > 80% folded in our simulation at 370 k , while , experimentally , the peptide is only 25% folded at this temperature . as with -helices , we expect ff15ipq to yield residue - specific propensities in -sheet stability , although the large difference in sequence between the two tested types of model -hairpins makes comparing them difficult . the aforementioned lack of a clear well in asp may destabilize the gb1 hairpin , which contains two adjacent asp residues , one of which forms part of the antiparallel -sheet and the other , the turn . the observed stabilities of chignolin and cln025 generally preclude the notion that ff15ipq is biased against the -sheet structure . further studies including additional hairpin sequences and parallel -sheet structures will be necessary to quantify and mitigate residue - specific biases for future ipolq force fields . in order to assess the stability of proteins with ff15ipq , we simulated the trp - cage miniprotein and a series of six globular proteins : bpti , villin , gb3 , ubiquitin , binase , and lysozyme . extensive experimental data are available for all of these model systems , providing excellent opportunities for validation of our simulations . we carried out 24 s of aggregate equilibrium simulations of trp - cage at temperatures ranging from 275 k to 325 k and simulations 210 s in duration of the six globular proteins at temperatures between 298 k and 303 k. details of our simulations are listed in table 1 . the designed miniprotein trp - cage is central to a long - running computational success story for amber force fields . folding simulations of this miniprotein using the amber ff99sb force field have successfully recovered the folded structure , yielded multiple folding and unfolding events , and provided a melting temperature ( tm ) of 318 k , which is in reasonable agreement with the experimental tm value of 315 k. as shown in figure 6 , trp - cage remained stable in our simulations between 275 k and 295 k , with an average backbone rmsd from the experimental nmr structure of < 1 , and unfolded between 305 k and 325 k. while our simulations are not extensive enough to obtain precise estimates of the tm , these results suggest that the tm value is somewhere between 295 k and 305 k , which is slightly lower than the experiment . each unfolding event is marked by an initial shift of the backbone / of pro 12 from the well to the ppii well , followed by the loss of the n - terminal -helical component of the polypeptide . notably , in our simulation that was run at 325 k , the protein refolded for 500 ns , indicating that the folded state is a stable free - energy minimum . thus , despite the extensive reoptimization of the parameter set , the important success of the amber force fields in modeling the stability of trp - cage has been maintained . stability of trp - cage over the temperature range of 275325 k over the course of 4-s simulations , as monitored by the backbone root - mean - square deviation ( rmsd ) , relative to the experimental nmr structure . note that the pro - rich sequence of trp - cage allows the opportunity to validate the unique pre - pro and terms of ff15ipq , because it contains gly , arg , and pro residues that precede pro . whenever trp - cage was folded in our simulations , gly 11 remained stably in its ppii well , while arg 16 was sampled broadly across the and ppii regions without a clear barrier between them . these results are in good agreement with the experimental nmr ensemble , within which the and backbone torsions of arg 16 are distributed in a line across these two regions , and with the pre - pro distributions observed for these residues in the ndrd dataset . also consistent with both the nmr ensemble and ndrd dataset are the observed distributions of pro 17 and pro 18 , which strictly maintained their positions in the ppii well , even as the protein unfolded . as shown in figure 7 , the overall structures of all six globular proteins bpti , villin , gb3 , ubiquitin , binase , and lysozyme remained stable over their entire simulations . the bpti protein remained closest to its crystal structure , yielding an average backbone rmsd of 0.7 , which may be a result of its three disulfide bonds among a total of 58 residues . all -helical and -sheet regions of this protein were retained for the entire simulation . the only notable deviation from the crystal structure of bpti was observed for ala 16 and arg 17 , which form the end of the loop preceding the first -sheet . these two residues , which occupy the and wells , respectively , in the crystal structure , made temporary excursions of several hundred nanoseconds to alternative conformations before returning to the crystal conformation ( figures s5 and s6 in the supporting information ) . the villin headpiece subdomain also remained close to its crystal structure , yielding an average backbone rmsd of 1.1 . in order to test our parameters for the noncanonical amino acid norleucine ( nle ) , which was included alongside the canonical residues during development of ff15ipq , we have simulated the fast - folding double - nle mutant of villin . the nle residues , both of which are located in the third -helix , strictly sampled the -helical well , suggesting that our parameters for nle are appropriate . the only significant deviation from the crystal structure of this mutant of villin is a 0.5 s excursion made by residues 1012 , which form the end of helix 1 and the loop linking helices 1 and 2 , to an alternative conformation different from that observed in the crystal structure ( figures s7 and s8 in the supporting information ) . stability of folded proteins over the course of 10-s simulations as monitored by the backbone rmsd , relative to the experimental structures . for binase , the mean rmsd relative to the ensemble of 20 nmr structures is shown , along with the range between the minimum and maximum values ( light blue shaded region ) . our simulation of gb3 yielded an average backbone rmsd of 1.0 from the nmr structure . three of the residues exhibit significant deviations from the crystal structure : leu 12 , asp 40 , and thr 55 ( figures s9 and s10 in the supporting information ) . the conformation of leu 12 , which is located in the turn linking the first and second -strand , falls precisely between the and ppii wells in the nmr structure , while both wells were almost equally sampled in our simulation . as a result of this increased conformational flexibility at leu 12 , adjacent residues also occasionally sampled conformations outside their nmr structure . the presence of a free - energy barrier between the and ppii wells is a necessity for maintaining stable conformations within these wells ; it is likely that the forces contributing to the stabilization of leu 12 s unusual conformation in the nmr structure are simply not captured by the functional form of ff15ipq . consistent with this hypothesis , almost - identical deviations were observed for the ff14sb force field that shares this functional form . the conformation of asp 40 , which is located in the loop between the -helix and third -strand , occupies the well in the crystal structure while other conformations , predominantly , were sampled in our simulations . indeed , based on this result and others presented below , the negatively charged residues of ff15ipq completely lack an well . as with leu 12 , the increased conformational flexibility of asp 40 led to broader sampling by adjacent residues . it is worth noting that gb3 contains two negatively charged residues , glu 15 and asp 22 , which remained stable in -sheets , indicating that the limited sampling of this region observed in our umbrella sampling simulations may be overcome within the context of a folded protein . finally , a notable deviation from the nmr structure occurs for thr 55 during the last microsecond of our simulation when the antiparallel -sheet hydrogen bonds between this residue and val 42 are broken , although the remainder of the -sheet remains in place . while this deviation may be a transient event , it could also be a consequence of the conformational deviations of the nearby asp 40 . similar to gb3 , ubiquitin in our simulations exhibited a low overall average backbone rmsd of 1.2 from the crystal structure , but significant deviations in certain regions . in particular , transient deviations from the crystal structure were observed for residues 811 , which form the turn connecting the first and second -strands ( see figures s12 and s13 in the supporting information ) . while these residues sampled conformations that were different from the crystal structure , their turn conformation was retained throughout 80% of the simulation , and experimental nmr relaxation data suggests that the region is truly flexible . a more significant deviation was observed for asp 52 and gly 53 , which are located in a loop region ; in the crystal structure , these residues both occupy the well , whereas , in our simulation , asp 52 and gly 53 shift to the ppii and wells , respectively . unlike the deviations observed in gb3 , this shift does not lead to broader sampling by adjacent residues or appear to otherwise destabilize the protein , suggesting that the observed alternative conformation may simply be erroneously modeled by ff15ipq to be lower in energy than the conformation found in the crystal structure . finally , after 8.5 s of simulation , glu 34 , which is the last helical residue in the central -helix , shifts to the ppii and regions , leading to shifts in residues 3336 . combined with the observations made for asp 52 , this shift suggests that , for negatively charged residues , ff15ipq may overstabilize ppii conformations , relative to . among the simulated globular proteins , the greatest deviations from the initial structure were observed for binase with an average backbone rmsd of 3.4 from the nmr ensemble of 20 models . the same average rmsd was also obtained , with respect to the crystal structure of wild - type binase , which differs in the amino acid sequence at six positions ( pdb code : 1gou ) . these larger differences are primarily caused by variability in loop regions , as noted for the experimental structures , and the core structure of binase remained relatively close to the experimental structures with an average backbone rmsd of 1.9 . the first loop , which consists of residues 3439 , adopted multiple conformations in our simulation , which is consistent with the nmr ensemble ( figures s15 and s16 in the supporting information ) . moreover , this loop adopts the conformation observed in the crystal structure for 75% of the simulation . the second loop consists of residues 5662 , which also sampled broadly in our simulations , consistent with diverse conformations in the nmr ensemble and poorly defined electron density in the crystal structure . notably , in both our simulation and the nmr ensemble , flexibility in this region extends to gly 67 . this difference may relate to the difference in sequence between the nmr and crystal structure proteins , in which ser 66 is replaced by gly and gly 67 by ser . the third loop , comprised of residues 7683 , is also broadly sampled in our simulation , and is the source of greatest difference , relative to the experimental structures . the final loop is comprised of residues 99104 and , in our simulation , we observe several residues sampling two different conformations , consistent with the observation of two states in crystal structures determined under different conditions . the largest protein system , lysosome , was simulated for 2 s , over which it exhibited an average backbone rmsd of 1.2 , with respect to the crystal structure . as shown in figures s17 and s18 in the supporting information , the largest deviations were found in the loop comprised of residues 100104 . residues 101104 adopted an alternative conformation , with the flanking residue val 99 no longer part of the preceding helix and gly 104 part of the following helix . similar to our observations for ubiquitin , this difference appears to be related to the shift of a negatively charged residue , asp 101 , from the well to the ppii well . bpti , gb3 , ubiquitin , and lysozyme have also been used for validation of previously developed force fields , including ff14ipq , ff14sb , and charmm36 . similar to these force fields , ff15ipq yielded low average backbone rmsd values for these proteins , relative to their initial structures ( i.e. , 1.2 ) . a key point to be considered while comparing our results to those of previous force fields is that advancements in gpu computing over the last several years have enabled us to validate ff15ipq using simulations up to 10 s , which is up to 10 times longer than those used for the other force fields . in particular , ff14sb was validated using sets of four 1-s simulations , while charmm36 was validated using 200 ns simulations . many of the key deviations that we observe do not occur for several microseconds ; for example , we observe changes in the c - terminus of gb3 after 9 s , and in the loops of ubiquitin after 8.5 s . these deviations are informative and will guide development of successors to ff15ipq , illustrating the utility of long - time scale simulations for force field development . a particularly appealing feature of the spc / eb water model with which we have developed ff15ipq is its ability to more accurately reproduce the rotational diffusion of solvated proteins relative to water models , such as tip3p and tip4p - ew . in order to measure how accurately the combination of ff15ipq and spc / eb are able to reproduce rotational diffusion , we branched off sets of ten 200-ns simulations in the microcanonical ensemble ( nve ) from our 10-s simulations in the canonical ensemble ( nvt ) for gb3 , ubiquitin , and binase , thereby avoiding perturbation of the dynamics by the use of a thermostat . as shown in table 3 , ubiquitin , gb3 , and binase diffused 14% , 15% , and 22% more slowly than that measured experimentally by nmr , respectively . note that the experimental values were corrected for differences in temperature , isotopic labeling , and solvent d2o content , potentially introducing error into the comparison of simulated and experimental values . interestingly , while the errors that we obtained were consistent with our test simulations with the amber ff99sb - ildn force field and spc / eb ( see figure s1 ) , we found charmm22 * and spc / eb to yield lower errors of 7% , 6% , and 16% , illustrating the coupling of solute and solvent parameters on the motions of proteins through solution . since the spc / eb water model had been empirically optimized for proteins with the amber ff99sb force field , this is no fault of ff15ipq ( or charmm22 * , for that matter ) , but suggests that improved performance might be obtained by optimizing the protein and solvent models in tandem . finally , it is worth noting that our use of the langevin thermostat in the nvt simulations results in 42%52% longer rotational diffusion times , relative to those of the nve simulations , demonstrating , for the first time ( to our knowledge ) , that the use of a thermostat can significantly perturb dynamical properties for proteins and not just for small molecules and polymer chains . uncertainties represent one standard error of the mean . experimental rotational diffusion measured using nmr relaxation and corrected for differences in temperature and d2o content between simulation and experiment . uncertainties represent one standard error of the mean calculated from 50 consecutive 200-ns blocks from a single 10-s simulation . uncertainties represent one standard error of the mean calculated from ten independent 200-ns simulations . a major advantage of performing simulations with accurate rotational diffusion is that one can directly calculate nmr relaxation parameters n r1 and r2 , and the n h heteronuclear noe , which report on the dynamics of individual residues , and compare these values with experiment . therefore , we calculated relaxation parameters for gb3 and ubiquitin , for which experimental data are available at five and four magnetic field strengths , respectively ( see figures s11 and s14 , respectively , in the supporting information ) . overall , our calculated r2 values are in excellent agreement with the experiment , with average mean absolute percent error ( mape ) values of 8% for gb3 and 9% for ubiquitin . our r1 values are also in good agreement , with average mape values of 10% and 12% , with a consistent offset observed across all residues . however , our heteronuclear noe values are somewhat poorer agreement , with average mape values of 22% and 30% for the two systems . residues for which our calculated r1 and r2 differ by > 20% from the experimental values may be due to limitations of our force field . notably , we find that several such residues are those for which we also observed deviation in sampled backbone conformations , relative to the experimental structures . for gb3 , leu 12 and asp 40 yielded above - average errors in r1 ( 25% ) , while gly 41 had a larger error in both r1 and r2 ( 47% and 33% ) . similarly for ubiquitin , lys 11 and asp 52 yielded above average errors in r1 ( 20% ) . also in ubiquitin , asn 25 yielded an error of 22% in r2 ; however , this residue is found experimentally to undergo chemical exchange , fluctuating at time scales beyond those captured by our simulations . among residues with errors below 20% , one particular trend is apparent : ile 7 , thr 16 , thr 49 , and thr 51 of gb3 and thr 12 and ile 36 of ubiquitin all yielded errors in r2 of 15% , despite having tightly restricted / sampling consistent with their experimental structures . this trend suggests that ff15ipq may have some discrepancy with the three branched residues that is not apparent when examining only backbone / preferences , and this will be the subject of further study . in order to test the suitability of ff15ipq for simulating disordered proteins , we focused on two model peptides : the n - terminal , 13-residue peptide fragment of the tumor suppressor p53 , and the 22-residue s - peptide fragment of rnase a. both of these disordered peptides ( p53 peptide and s - peptide ) only adopt -helical conformations when bound to their structured partner proteins ( mdm2 and s - protein , respectively ) . for each of these peptides , we carried out two 10-s simulations : one of the isolated peptide and the other of the native peptide both peptides in their isolated states adopted conformations distant from their partner - bound conformations , sampling a diverse set of conformations with average backbone rmsds of 5 from their corresponding bound conformations in the crystal structures of the peptide protein complexes and maintaining only 25% of their native intrapeptide contacts . furthermore , the p53 peptide only transiently adopted -helical conformations that resembled its partner - bound conformations ( backbone rmsd value of <3 ) with these conformations unfolding within 200 ns ( see figure s19 in the supporting information ) . in contrast , the s - peptide did not even transiently sample -helical conformations resembling its bound state ; instead , the peptide formed -hairpins that persisted for periods as long as 4 s ( see figure s22 in the supporting information ) . stability of p53 and s - peptide alone and in complex with binding partners mdm2 and s - protein over the course of 10-s simulations as measured by backbone rmsd relative to ( a , b ) the crystal structures and ( c , d ) the percent of native contacts formed . in our simulation of the p53/mdm2 complex , the peptide remained stably bound to its partner protein for the entire 10 s of simulation , with an average backbone rmsd of 1 from its bound conformation in the crystal structure ( figure 8) . curiously , while most of the intramolecular native contacts of the p53 peptide and mdm2 were retained ( 95% and 85% , respectively ) , only 60% of the intermolecular p53/mdm2 native contacts persisted . examination of the structure showed that many of these contacts lay just below the threshold distance of 5.5 between their heavy atoms in the crystal structure . thus , these contacts were no longer formed when slightly different conformations were adopted in our simulations . throughout our simulation , the p53 peptide retained the -helical structure of residues 1925 ( recall figure s19 ) . in contrast , our simulation of the s - peptide / s - protein complex sampled conformations more distant from its crystal structure , with average backbone rmsd values of 3 for both s - peptide and s - protein . although the s - peptide partially lost its -helical structure near the n - terminus from 1.5 s to 5 s in our simulation ( see figure 8 and figure s22 ) , the structure reformed and persisted for the remainder of the simulation during which both the s - peptide and s - protein retained most of their intramolecular native contacts ( 90% and 80% , respectively ) . similar to the p53/mdm2 complex , the s - peptide / s - protein complex retained 60% of its peptide protein contacts throughout our simulation . our simulations of these flexible , disordered peptides provide an additional opportunity to validate the backbone conformational preferences of ff15ipq . from our four simulations , we have calculated the backbone / sampling of the p53 peptide ( figure 9 ) and s - peptide ( figure s23 in the supporting information ) . for comparison , we have provided the distributions for the peptide sequences obtained from the ndrd dataset , which accounts for the influence of adjacent residues on each distribution . in our simulation of the p53/mdm2 complex , residues 1827 of p53 occupied exclusively the wells observed in the crystal structure , aligning with the observed low rmsd and high percentage of native contacts . our simulation of the isolated p53 peptide exhibits similarities with the ndrd distribution for most residues , but several inconsistencies are informative : in particular , the neutral residues exhibit reasonable overall agreement and the bulky aromatic residues phe 19 and trp 23 closely resemble the ndrd distributions , while leu 22 and leu 25 sampled of the -region more extensively than suggested by the ndrd . the negatively charged residues glu 17 and glu 28 sampled consistently with the ndrd , while asp 21 missed sampling in the region . the sole positively charged residue , lys 24 , sampled the -well more backbone conformational sampling of the disordered p53 peptide observed in 10-s simulations ( a ) in complex with the mdm2 protein and ( b ) alone . for comparison , panel ( c ) shows distributions for the p53 sequence obtained from the neighbor - dependent ramachandran distribution ( ndrd ) dataset , derived from conformations observed in the loops of crystal structures . in our simulation of the s - peptide / s - protein complex , the crystal conformations were retained for most of the simulation ( see figure s23 ) . as described above , several residues near the n - terminus left the -helical well , and the adjacent thr 3 , which is not helical in the crystal structure , eventually joined the helix as it reforms . the clearest difference from the crystal structure is found for asp 14 ; this residue occupies the well in the crystal structure , which is not present for asp in ff15ipq , causing it to adopt a ppii conformation . within the s - protein , conformation ( 100 , 130 ) , which was retained throughout our simulation ( figure s25 in the supporting information ) . in our simulation of the isolated , unbound s - peptide , the formation of long - lived -hairpin structures prevented us from obtaining converged conformational preferences for comparison with the ndrd distributions , despite the long duration of the simulations ( 10 s ) . taken together , the above results indicate that ff15ipq can reliably predict disorder as well as order for peptides that fold upon binding their partner proteins . these encouraging results are worth pointing out since ff15ipq was not specifically parametrized for disordered peptides / proteins , as is the case for contemporary force fields such as ff03w and its subsequent variants . as shown in figure 1 , both ff03 ( whose atomic charges and radii are shared by ff03w ) and ff15ipq are able to reliably model propensities of salt - bridge formation , which can be critical for such systems that are rich in polar and/or charged residues . thus , ff15ipq is a reasonable alternative to ff03w for the simulation of disordered peptides / proteins . since the establishment of the one atom , one site model of all - atom fixed - charge force fields over 20 years ago , several major lineages of protein force fields and countless branches have been developed through cycles of validation and refinement . in this work , we present the ff15ipq force field , the latest in the amber ipolq lineage , and validate its accuracy by > 200 s of aggregate md simulation . the distinguishing features of ff15ipq are ( i ) a charge set that accounts directly for water induced polarization , ( ii ) the incorporation of two related charge sets for creating new force fields based purely on ab initio calculations , ( iii ) the scope of the parameter optimization , including backbone angles alongside torsions , and ( iv ) the degree of automation and transferability of the methods to other regions of chemical space . our simulations suggest that ff15ipq yields reasonable salt - bridge propensities , maintains secondary structures and globular protein folds on the s time scale , predicts order as well as disorder in protein structures , and yields strong agreement with nmr j - couplings and relaxation rates . however , even with this extensive amount of validation , several unconverged results remain and will be explored further using enhanced sampling techniques such as replica exchange or recent variants of the weighted ensemble path sampling strategy . a major motivation for creating ff15ipq was to address concerns about the overstabilization of salt - bridge interactions by ff14ipq , which is a limitation that is shared by other contemporary force fields . we addressed these concerns by abandoning the mixed lennard - jones radii of ff14ipq and instead increasing the radii of polar hydrogen atoms bonded to nitrogens in both the protein backbone and side - chains to yield more accurate salt - bridge propensities . the atomic charges of the force field were then rederived by applying the automated machinery , which had created ff14ipq , exchanging the tip4p - ew water model for spc / eb . finally , all torsion and selected backbone angle parameters were refit to a qm dataset over four times as large as that used for ff14ipq . in doing so , we found that angle optimization was essential for recovering reliable results with our more - extensive dataset ( see the timeline of development while the angle optimization feature and other particulars of the torsion fitting are subjects of ongoing development in our force field engine mdgx , our results with ff15ipq indicate that the workflow illustrated in figure 10 is a viable approach to creating new force fields . each step entails additional layers of details in order to address practical considerations such as infinite electrostatics or the forms of molecular mechanics basis functions and the shape of the target energy surface . we have addressed each of these issues in the theory and methods sections as well as in prior publications , but the synthesis of all these details reflects the physical arguments behind the ipolq charge derivation . starting from an existing model , selected global changes are optionally first applied to obtain an initial model for optimization . the ipolq charge deviation protocol is then used to fit a pair of atomic charge sets for the vacuum ( qvac ) and solution ( qsolv ) phases . the vacuum - phase charges are used to fit parameters for bonded terms to vacuum - phase qm targets , and these parameters are subsequently paired with the solution - phase charges to yield a complete force field for solution - phase simulations . the force field is then validated through extensive md simulation , informing future development . this approach should be viewed in context with the contemporary force balance approach , which also performs sweeping optimization of hundreds of parameters simultaneously . unlike our mm - minimized conformations , force balance typically considers conformations qm - minimized at the same level of theory at which the target qm energies are calculated , although this is not a strict requirement . going beyond the capabilities of mdgx , force balance includes numerous nonlinear optimization methods and offers the capability to incorporate results from sources beyond qm single - point energies , including in vitro experiments , directly into the parameter optimization . in the future , such diverse targets might be paired with the ipolq method , for example , by using the vacuum charge set ( qvac ) for comparison with vacuum - phase qm data , but using the polarized charge set ( qsolv ) in simulations for comparison to experimental results in solution . it is rather remarkable that a viable protein force field can be produced within months , almost entirely from qm data . also noteworthy is the fact that features such as angle optimization and generational refinement , which had incremental but definite effects on the accuracy of data fitting , could be so influential in the final result . one way of considering the remaining error in our mm model is to partition it between two sources : bonded and nonbonded interactions . the improvements in ff15ipq that were obtained relative to ff14ipq , whose nonbonded parameters are of similar accuracy , resulted from optimization of angles and the branching of bonded parameters . while the inclusion of anharmonicity in bond and angle stretching or a spline - based treatment of torsion cross terms ( cmap ) may reduce errors further , greater improvements might be accomplished in the nonbonded interactions . for this reason , the next planned advance of the amber ipolq force field lineage is to improve the accuracy of electrostatic interactions by making liberal use of virtual charge sites . paradigm used for ff15ipq , which was established several decades ago , appears sufficiently accurate for most purposes , economical by construction , and thoroughly optimized in existing md engines . models with significant numbers of virtual charge sites are presently in the process of becoming established . these models offer improved accuracy for various chemistries with clear physical motivations , accompanied by a more modest increase in computational cost than that afforded by polarizable functional forms . future amber ipolq development at the one - site - per - atom level will continue to explore the applicability of our methodology to the chemical space of other biologically important molecules , including nucleic acids , carbohydrates , and small molecules . further ahead along the path lie multisite ipolq models , which will push the mimicry of qm potential energy surfaces within the confines of a nonpolarizable model to new levels .

we present the amber ff15ipq force field for proteins , the second - generation force field developed using the implicitly polarized q ( ipolq ) scheme for deriving implicitly polarized atomic charges in the presence of explicit solvent . the ff15ipq force field is a complete rederivation including more than 300 unique atomic charges , 900 unique torsion terms , 60 new angle parameters , and new atomic radii for polar hydrogens . the atomic charges were derived in the context of the spc / eb water model , which yields more - accurate rotational diffusion of proteins and enables direct calculation of nuclear magnetic resonance ( nmr ) relaxation parameters from molecular dynamics simulations . the atomic radii improve the accuracy of modeling salt bridge interactions relative to contemporary fixed - charge force fields , rectifying a limitation of ff14ipq that resulted from its use of pair - specific lennard - jones radii . in addition , ff15ipq reproduces penta - alanine j - coupling constants exceptionally well , gives reasonable agreement with nmr relaxation rates , and maintains the expected conformational propensities of structured proteins / peptides , as well as disordered peptides all on the microsecond ( s ) time scale , which is a critical regime for drug design applications . these encouraging results demonstrate the power and robustness of our automated methods for deriving new force fields . all parameters described here and the mdgx program used to fit them are included in the ambertools16 distribution .

Introduction Theory Methods Results Discussion

PMC4411584

acute spinal cord injury ( sci ) is a potentially devastating occurrence that affects 270,000 people each year in the united states and is expected to increase . recovery from sci is highly variable and felt to be associated with a multitude of factors including the care rendered during the acute hospital setting . one such intervention is raising the blood pressure to improve blood flow to the penumbra of tissue near the injury site that is at risk of edema and infarction . this risk is accentuated by impaired vascular reactivity and loss of auto - regulation of the injured spine , making it vulnerable to systemic hypotension and impaired functional recovery . raising the blood pressure is often accomplished with volume resuscitation and vasopressor support ; however , there is no gold standard in terms of optimal perfusion . there have been several published studies on the effect of blood pressure management in acute sci , the largest of which contained 103 patients , 41 of which required vasopressors . despite this , there only exists level iii evidence suggesting a target mean arterial pressure ( map ) goal of 85 - 90 mmhg . the most tangible outcome of improved spinal cord perfusion is ultimate functional neurologic outcome . in this current study , we hope to assess the largest published cohort of acute sci patients and evaluate functional outcome as it relates to set map goals during hospitalization . we hypothesize that increased spinal cord perfusion will not be associated with improved functional outcome by hospital discharge . spinal cord perfusion will be measured using both map and number of episodes of hypotension during the initial hospitalization . this single institution study was conducted at a level 1 trauma center and regional sci center ( one of 14 such centers designated by the national institute on disability and rehabilitation research ) . with institutional review board approval , a retrospective review of the prospectively - entered trauma database was performed to identify all patients with an acute cervical or thoracic sci during a 2.5-year period , january 2007 through june 2009 . the hospital 's electronic medical record was then queried for lowest and average map for each hour of the initial 72 h of hospitalization . this allowed for quantification of mean map and the total number of episodic relative hypotensive events ( maximum 1/h ) . these relative hypotensive events were subsequently denoted by the creation of theoretic map set points ( > 90 , > 85 , > 70 , > 65 mmhg ) for which the data were compared . at each map set point , patients were divided into a quartile distribution based on the number of relative hypotensive events . division into quartiles and not using the absolute number of events was necessary as the number of events differed significantly based on the theoretic map goal set point . severity of sci and functional improvement was assessed using the american spinal injury association 's ( asia ) motor score ( ams ) . ams is more reliable than the more popular asia grade because it allows for each limb to be scored separately increasing its sensitivity and has thus been found to be the best indicator for observing the effects of therapeutic interventions . ams is calculated by assessing function in 5 key muscle groups per extremity , each muscle group with a maximum score of 5 , creating a total body maximum score of 100 . american spinal injury association motor score data were abstracted from the daily progress notes of the physical medicine and rehabilitation ( pm&r ) physicians . in a similar fashion to mean , map and number of relative hypotensive episodes , admission ams and the change in ams during the hospitalization was assessed in quartile fashion using the same theoretic map goal set points . finally , the need for vasopressor support during the initial 72 h of hospitalization was also recorded for each patient . statistical analysis was performed using spss version 20.0.0 ( spss , inc . , 2011 , all data were subjected to descriptive statistical analysis yielding frequency scores for categorical data and mean scores with standard deviations for continuous / interval data . statistically significant differences were evaluated by conducting inferential statistical analysis using the chi - square test or fisher 's exact test for categorical variable comparisons and the student 's t - test or analysis of variance ( anova ) for continuous / interval data comparisons . bonferroni correction was used to compare the number of patients in one quartile with the other quartiles while running the anova . a p 0.05 was considered as statistically significant . two hundred and twenty - three patients presented with acute cervical and thoracic sci during the study period . figure 1 displays those patients who were excluded from data analysis for missing telemetry data , ams documentation , or missing charts . sci : spinal cord injury , map : mean arterial pressure , ams : asia motor score when patient telemetry data were subjected to theoretic map set points , the number of episodes below that set point was calculated . patients were then grouped into quartiles for statistical analysis based on the number of episodes . table 1 displays the results of the set points 90 , 85 , 70 , and 65 mmhg . at the 90 and 85 mmhg set points , a statistically significantly higher number of relative hypotensive episodes related to lower admission ams ; however , there was no related difference in the change in ams during the acute hospitalization . this difference was no longer statistically significant at lower theoretic map set points ( 70 and 65 mmhg ) . the effect of admission ams and change in ams during hospitalization on the number of episodes of hypotension ( stratified by quartile ) ; further stratified by theoretic map goal set - point table 2 displays patient demographics , mechanisms of injury , and injury level . it then further segregates data by improvement or worsening of ams during the acute hospital admission . table 3 displays the need for vasopressors and correlates that with the ams at admission change in ams during hospitalization , and the number of hypotensive episodes at each theoretic map goal set point . results revealed the need for vasopressors correlated with the number of hypotensive episodes and inversely related to admission ams at all theoretic map goal set points , but was not correlated with the change in ams during the hospitalization . patient demographics and clinical features with comparison of those whose ams improved vs. those whose worsened during the admission the effect of the admission ams , change in ams during hospitalization and the number of hypotensive episodes by theoretic map goal set - point versus the need for vasopressors our results showed lower maps , more frequent hypotensive episodes , and an increased need for vasopressors occurred in patients with more severe injuries . these findings are significant because contemporary clinical practice in sci still includes driving map goals to supra - physiologic levels . whereas in true vasoplegia , where these agents improve outcome despite side - effects , there use to create relative hypertension alters this risk / benefit ratio . further , over resuscitation with volume can result in fluid overload that has been associated with both cardio - pulmonary complications and surgical complications . thus , deploying these strategies warrants appropriate considerations of the deleterious effects and determining the associated risk / benefit ratio . fortunately , approximately 60% of acute scis are incomplete , a percentage that has increased in the last 15 years . this select population was chosen secondary to the greater likelihood of autonomic dysregulation from loss of normal sympathetic function . lumbar sci patients are theoretically less likely to benefit from induced hypertension . having conducted this study at a regional sci center , the study population is likely biased toward more severe injuries that community practitioners felt were too complex . these more severe injuries may have biased our results but at the same time represent the most at - risk population . the lack of support for increasing map goals in our results is despite the fact that not only does our center practice induced hypertension , but it also has all of the collaborative resources of a regional sci center . this includes the multidisciplinary coordination of care between trauma , orthopedics , neurosurgery , pm&r , amongst others . ams improvements can , therefore , be noted during the acute hospitalization , but also at rehab and beyond . our dataset was limited to the acute hospitalization , but future studies of more delayed outcomes may yield different findings . perhaps some patients normally require a hypertensive state due to chronic vascular disease and after injury are relatively hypotensive as compared to their baseline . this is well described in hypertensive end - stage renal patients who become transiently relatively hypotensive during dialysis and develop cardiac ischemia . in our study , we did not look at preexisting hypertension but this may be a more select population to study for the benefits of increased map goals after sci . another limitation of the current study is the fact that we only looked at the lowest map during a 1 h period , but we do not have a record of how long that hypotensive episode lasted . in theory , this limitation should affect both groups equally but this can not be measured within the limits of our retrospective dataset . finally , a single institution and retrospective nature of this study are also noted limitations . the frequency of episodes of relative hypotension and the need for vasopressors are progressively related to more severe sci , as denoted by lower admission ams . however , episodes of hypotension and the need for vasopressors did not affect the change in ams during the acute hospitalization , regardless of theoretic map goal set - point . arbitrarily elevated map goals may not be efficacious but further prospective study with longer - term follow - up is needed .

introduction : acute spinal cord injury ( sci ) is often treated with induced hypertension to enhance spinal cord perfusion . the optimal mean arterial pressure ( map ) likely varies between patients . arbitrary goals are often set , frequently requiring vasopressors to achieve , with no clear evidence supporting this practice . we hypothesize that increased map goals and episodes of relative hypotension do not affect hospital outcome.materials and methods : all cervical and thoracic sci patients treated at a level one trauma and regional sci center over at 2.5-year period were retrospectively reviewed . lowest and average hourly map was recorded for the first 72 h of hospitalization , allowing for quantification of mean map and the total number of episodic relative hypotensive events . these data were further compared to daily american spinal injury association motor score ( ams ) , which was used to determine the severity of sci and improvement / decline during hospitalization . patient 's data were finally analyzed at theoretic map set points.results:one hundred and five patients had complete data during the study period . at higher theoretic map set points ( 85 and 90 ) , increased number of relative hypotensive episodes correlated with lower admission ams ( 85 mmhg : < 10 episodes , ams 66.2 ; > 50 episodes , 22.0 ; p < 0.001 ) and the need for vasopressors ( p < 0.03 ) but showed no statistical change in ams by hospital discharge . the need for vasopressors correlated with the number of hypotensive episodes and inversely related to admission ams at all theoretic map goal set points but was not correlated with the change in ams during the hospitalization.conclusions:the frequency of relative hypotension and the need for vasopressors are progressively related to more severe sci , as denoted by lower admission ams . however , episodes of hypotension and the need for vasopressors did not affect the change in ams during the acute hospitalization , regardless of theoretic map goal set - point . arbitrarily elevated map goals may not be efficacious .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC5014240

the heat energy that is dissipated by auroral currents has a significant influence on variations in the neutral density and temperature in the thermosphere . the flux of solar ultraviolet radiation is the dominant source of the heating of the thermosphere when geomagnetic activity is low [ tobiska et al . , 2008 ] ; at times of high activity the more important component is the additional energy that is dissipated by the highlatitude electric fields and currents . this energy source is driven by the solar wind and interplanetary magnetic field ( imf ) that originate from the sun . at times of moderate driving the ionospheric heating in both hemispheres totals several hundred gigawatts ( gw ) [ chun et al . , 1999 , 2002 ; the auroral heating may exceed a thousand gw , resulting in neutral density changes that increase the drag on objects in low earth orbit , to the extent that orbit prediction and tracking becomes difficult [ lu et al . it has become apparent that the amount of nitric oxide ( no ) in the thermosphere has a significant influence on the neutral temperature and density . the chemical reactions that produce no have a strong temperature dependence [ kockarts , 1980 ; bailey et al . , 2002 ; barth et al . , 2009 ; barth , 2010 ] , so that heating within the ionosphere enhances the level of no in the lower thermosphere . due to the sensitivity of no production to temperature , highlatitude joule heating has a significant role , as noted by barth and others . auroral electron precipitation also has a significant role in no production , mainly by increasing the number density of n(d ) and n(s ) atoms in the lower thermosphere , which react with oxygen molecules to produce no [ siskind et al . , 1989 ; that the total energy deposited by the electrons tends to vary in proportion to the total joule heating . the ratio of joule / particle heating in the events studied ranged from 1.7 to 6.9 , with a mean value of 2.8 . at low latitudes , particularly when geomagnetic activity is low , soft xray radiation from the sun is the primary source of no production [ barth et al . , it is known that optical emissions from nitric oxide are efficient in cooling the thermosphere [ kockarts , 1980 ; sharma et al . the result is that no acts as a selfregulating , natural thermostat for the thermosphere that reduces the effects of the auroral disturbances [ mlynczak et al . , 2003 , 2005 ] . lu et al . had examined the relationship between global joule heating and global radiative cooling emissions due to no . two models were coupled together in their study to derive the joule heating energy input as well as model the no energy output , during six geomagnetic storm periods . firstprinciple numerical simulations from the thermosphere ionosphere electrodynamic general circulation model [ richmond et al . , 1992 ] were coupled with the assimilative mapping of ionospheric electrodynamics ( amie ) data assimilation algorithm [ richmond , 1992 ] . the numerical results were compared with measurements of optical emissions from no , obtained at a wavelength of 5.3 m by the sounding of the atmosphere using broadband emission radiometry ( saber ) instrument on the thermosphere ionosphere mesosphere energetics and dynamics ( timed ) spacecraft . they found strong correlations between the global no power and timelagged joule heating during the selected events . used their results to derive a predictive formula for the no cooling power , based on the f 10.7 and k p indices . another formula had used solar irradiance measurements from the solar euv experiment , also on the timed satellite , rather than the f 10.7 flux . compared with the saber measurements , these prediction formulas had correlation coefficients 0.89 and 0.90 . computed the total poynting flux into the northern and southern polar regions with an empirical model and compared these results with changes in the global exospheric temperatures . these temperatures were derived from neutral density measurements on the challenging minisatellite payload ( champ ) and gravity recovery and climate experiment ( grace ) satellites [ tapley et al . a differential equation was developed for calculating changes in the global exospheric temperature as a function of time . the formula required only the total poynting flux into the ionosphere , derived from imf measurements . the ionospheric heating increases the temperature of the thermosphere , and in the absence of further heating the temperature decreases at an exponential rate . it was found that the exponential cooling rate was not always the same . following time periods that had high levels of ionospheric heating and greater temperature increases , the thermosphere was observed to cool off faster than when the heating levels were more moderate . in order to obtain consistently good results in matching the differential equation model to the champ and grace measurements , weimer et al . an additional variable was added to the equations , which increased in proportion to the amount of heating and decreased when heating was absent . this variable lowered the value of the exponential time constant , resulting in a more rapid cooling of the thermosphere . all factors in the equations were derived by an automated , iterative fit to the density measurements . the enhanced thermosphere cooling was assumed to be due to enhancements in the concentration of nitric oxide , but this conjecture was not proven at the time . one purpose of this paper is to verify the results obtained by weimer et al . second , on the basis of new information provided by this comparison , the thermosphere temperature calculation has been improved . a third objective of this paper is to demonstrate exactly how sensitive the nitric oxide levels are to auroral heating and the thermospheric temperature and show their high level of correlation over long time periods . the saber instrument is a broadband radiometer that measures infrared radiance in 10 bands between 1.27 and 15 m . emissions from carbon dioxide ( co2 ) are measured in addition to the no . the instrument scans the earth 's limb from approximately 400 km tangent altitude down to the surface , recording an infrared radiance sample every 0.4 km . approximately 1400 profiles of infrared emission are obtained for each of the 10 saber channels in 1 day . the nominal instantaneous field of view of the instrument is 2 km at the tangent point on the earth 's limb . the measured radiances are converted to volumetric emission rates in w m , and each scan is then integrated vertically giving fluxes of power in w m. the fluxes for each day are integrated in latitude and longitude to give a daily global power emitted by the entire atmosphere , in watts . further details about the instrument and the data processing procedures are provided by mlynczak et al . a standard data product provided by the saber science team is a text file that provides the total global power radiated by no and co2 on a daily basis . figure 1 shows the daily power from no for 8 years of data , from 2002 to 2010 . as is evident in this figure , the radiated power follows the solar cycle , with the declining phase of solar cycle 23 evident , in addition to approximately 27 day activity variations due to solar rotation . even more apparent are large increases in radiative power at the times of large geomagnetic storms . global radiated power due to nitric oxide , measured with the saber instrument . the daily mean radiative power integrated over 100200 km , in units of gigawatts ( gw ) , is shown for years 2002 through 2009 . while the total power of the co2 emissions greatly exceeds that of the no emissions , as illustrated by mlynczak et al . , they come from a region that is mainly at or below the base of thermosphere , and they are not as sensitive to variations in the solar cycle and solar activity . the co2 cooling rates are not included in the work described here because they have a strong semiannual variation that has no correlation with the no cooling or geomagnetic activity . as mentioned in section 1 , weimer et al . had compared heating in the polar regions with changes in the global exospheric temperatures that were obtained from the champ and grace satellites . the total poynting flux into both hemispheres was calculated with the method described by weimer [ 2005a ] , using for input values the imf and solar wind measurements from nasas advanced composition explorer ( ace ) satellite . the magnetic field instrument [ smith et al . , 1998 ] provided the imf , while the solar wind velocity and number density are from the solar wind electron , proton , and alpha monitor instrument [ mccomas et al . , the level 2 data from the nasa archives are used , available at ftp://cdaweb.gsfc.nasa.gov/pub/data/ace . the most recent version of the model described by weimer [ 2005b ] was used , referenced here on as the w05 model . the poynting flux is derived from the electric potential and fieldaligned current mappings in this model , which provide both the electric field and magnetic field perturbations above the ionosphere . this method does not depend on any model of the ionospheric conductivity , as the conductivity variations , as a function of dipole tilt angle and location , are implicitly included in the measurements used for the model 's development . the total poynting flux flowing into both polar hemispheres as a function of time was compared with measurements of neutral densities in the thermosphere at two altitudes , obtained from accelerometers on the champ and gracea satellites . the derivations of the density data used in the calculations are described by sutton et al . and were obtained from an online database ( http://sisko.colorado.edu/sutton/data.html ) at the university of colorado . 2008 ] was used to convert the neutral density values to a measurement of thermosphere temperature . this model derives all global densities from a parameter that jacchia refers to as the global nighttime minimum exospheric temperature , and referred to as t c. the same notation was employed by bowman et al . and is also used here for continuity rather than the more descriptive symbol tmin . in the jb2008 model , the maximum exospheric temperature on the dayside is always 1.3 times the value of the minimum , t c. daily values of t c in jb2008 are obtained from indices of solar radiation using this formula : ( 1)tc=392.4 + 3.227fs+0.298f10 + 2.259s10 + 0.312m10 + 0.178y10where f 10 is a proxy index for the 10.7 cm solar radio flux . the symbols s 10 and m 10 represent proxy indices for the extreme ultraviolet ( euv ) flux measured on the soho satellite and the medium ultraviolet flux from noaa satellites . the y 10 index is a weighted mixture of xray and lyman , with the xray measurements , at wavelengths of 0.10.8 nm , coming from a spectrometer instrument on the goes satellite , and lyman from the timed satellite . the derivation of these indices , as well as documentation of the data sources , is described by tobiska et al . . the shortened notation used here follows from the same convention employed by bowman et al . . the delta values in ( 1 ) represent the difference of the daily and 81 day centered , boxcar average value of each index , and fs is obtained from a weighted combination of the runningaverage values of f 10 and s 10 . more rapid changes to the t c temperature , due to the auroral heating associated with geomagnetic activity , are referred to as t c. in the jb2008 model hourly values of the t c correction are calculated from the d s t index , using the formula by burke . had developed a method to derive measurements of t c from the champ and grace data , using the jb2008 model in reverse , to find the temperature value that produced a match with the measured neutral densities . the orbitaveraged values of this t c from the two satellites were in very close agreement , even though they are at different altitudes and often in different orbit planes . it was found that t c could be predicted very well from the poynting flux in the w05 model with this formula : ( 2)tc(tn+1)=tc(tn)tc(tn)(t/c)+hj(tn)t ( 3)no(tn+1)=no(tn)no(tn)(t/no)+hj(tn)t ( 4)c=14.6(h)0.281no the h j in this formula is the total joule heating from the w05 model in both hemispheres , and is a scaling factor that relates the temperature increases to the heating input within each time step , having a value of 6.910k/(gw min ) or 1.210 k / gj . the exponential cooling rate of the thermosphere is c , and no represents the changes in nitric oxide concentration , using an arbitrary scaling that has no physical units assigned . equation ( 3 ) causes this no level to increase in proportion to the heating , with scaling factor , and decay with an exponential time constant no = 28.0 h. the value of was preset to a value that increased the level of no by 0.0001 for each gw of heating , in every 4 min time step . as equation ( 4 ) shows , as the level of no increases , then the time constant c for the thermosphere cooling rate becomes smaller , resulting in a more rapid exponential decay of the temperature . the values of all constants other than were determined by fitting the calculated values of t c to the values that were derived from the champ and grace density measurements . all data from the years 2003 through 2005 were used for this least error fit . the downhill , simplex method described by press et al . was used in a program that reiteratively adjusts all variables until a minimum value of the error is reached , to within the limits of assigned tolerance values . a modification to their original program was made such that the tolerance is specified as a vector rather than a scalar . one other adjustment was required in order to obtain the best fit , that being an additional saturation factor for the total heating from the w05 model . the ionospheric electric potentials are known to increase , then rolloff to a nearly flat slope , as the magnitude of the interplanetary electric field increases [ siscoe et al . , , there were no data at sufficiently high driving levels to fully resolve the rolloff of the electric potentials at large imf magnitudes . in this study it was found that an additional , nonlinear reduction in heating at high driving levels was needed in order to produce good fits at both low and high levels of heating , otherwise the calculated temperatures were too large in the more extreme events . this additional saturation was provided by ( 5)jh = jw051 + 50039001.+jw053900which maintains a nearly linear curve with slope of 1 up to 500 gw before the rolloff toward saturation . the value of 3900 that produced the best fit was found with a reiterative process . an example of the results from the t c calculation in ( 2 ) , ( 3 ) , ( 4 ) , ( 5 ) is shown in figure 2 for the entire year 2003 . the red line in figure 2a shows the total heating from the w05 model that is calculated from the ace measurements in the solar wind , while the red line in figure 2b shows the t c values that are calculated . the superposed black line in figure 2b shows the t c values derived from the grace density measurements . for the majority of the time these two curves are in good agreement , with the most obvious difference being at the end of october 2003 , during the wellknownhalloween storm . in this event the thermosphere had cooled so rapidly that the measured value of t c went negative , meaning that the temperature went below the more slowly varying value of t c derived from the solar indices by application of ( 1 ) . this rapid cooling phenomenon during this event has previously been noted by weimer et al . and lei et al . . results from the thermosphere temperature calculation described by weimer et al . , for 2003 . ( a ) the total auroral heating , in gw , calculated from the w05 empirical model . ( b ) black line shows the t c parameter derived from neutral density measurements on the grace satellite . the red line shows t c that are derived from the heating , using equations ( 2 ) , ( 3 ) , ( 4 ) . ( c ) the blue line shows a variable that is proportional to changes in global no levels , from ( 3 ) . the superposed blue line shows the linear fit of the values shown in figure 2c , after 24 h smoothing , from equation ( 6 ) . the value of the no variable calculated with ( 3 ) is illustrated with the blue line in figure 2c . for comparison , the saber measurements of the no radiated power during this time period are shown in figure 2d with the green line . these data are the same as shown in figure 1 . for an easier comparison the values of no computed at 4 min intervals have been smoothed to the same 24 h cadence of the saber data and fit to the measurements . the blue line in figure 2d shows the result of the fit , with the linear equation ( 6)nosaber=33 + 29no3no3 refers to the values computed with ( 3 ) , and the correlation with nosaber is 0.88 , with a mean error ( absolute deviation ) of 38 gw and a standard deviation of 53 gw . higher correlations were obtained in other years . while equations ( 2 ) , ( 3 ) , ( 4 ) do a good job of modeling the temperature perturbations that were derived from the density measurements , there is one deficiency in that the t c value from ( 2 ) can not be negative . on the other hand , the measured values of t c this discrepancy causes some inaccuracy in the results in situations where accuracy is needed the most . the comparison with the saber measurements confirms that the no radiated power does indeed increase in proportion to the total auroral heating . this validation , coupled with a desire to obtain t c values that can be negative , leads to the realization that the calculation could be modified so that an increase in the level of no leads to a direct reduction in the temperature due to radiated power rather than an ad hoc reduction in the cooling time constant . the revised equation for calculating t c is ( 7)tc(tn+1)=tc(tn)tc(tn)t/c+hj(tn)tcno(tn)t in this equation c no represents cooling from the nitric oxide that contributes to a temperature reduction in each time step , while the fixed time constant c controls the exponential decay due to heat conduction and other radiative species . if the value of t c at time step t n does become negative , then the second term , with the time constant c , on the right side of ( 7 ) is not included . the value of a dimensionless quantity no with arbitrary scaling is calculated from the heating as before : ( 8)no(tn+1)=no(tn)no(tn)t/no+hj(tn)t as in the previous equation , the value of was preset to a value that increased the level of no by 0.0001 times the total heating in gw , in every 4 min time step . the value of c no is proportional to the value of no from ( 8) times the total temperature , minus an offset : ( 9)cno(tn)=no(tn)(tsolar(tn)+tc(tn) ) t solar represents the variations in the exospheric temperature due to solar radiation , as calculated with ( 1 ) . it is noted that how no is used in ( 9 ) is quite different from how it acts in ( 4 ) ; this symbol represents a different quantity . the variables in these equations were derived from a least error fit with the t c values that were obtained from the grace neutral density measurements over a 3 year time span . as before , the downhill , simplex method [ press et al . , 1986 ] was used for the fit . the revised fitting process now included a simultaneous adjustment of the saturation curve in ( 5 ) , increasing the number of variable parameters to six . since the downhill method requires an initial starting guess for the solution , it was helpful to first narrow down the approximate location of the solution in the sixdimensional space , through a trial of all combinations on a coarse grid . the champ data were not used in this fit , due to some concerns about the precision of the drag calibrations . the result of the new fit with equations ( 7 ) , ( 8) , ( 9 ) was = 7.0810 k/(gw min ) ( 1.210 k / gj ) , c = 16.3 h , = 3.9510/min , no=14.9 h , and = 544k . the revised heating saturation curve had 1560 in ( 5 ) , rather than 3900 , resulting in a greater reduction in the heating at high driving levels . figure 3 shows the result of the new t c calculations for the same time span as shown in figure 2 . as the heating from the w05 model is exactly the same , it is not included in this graph . the plot in figure 3a has the t c levels from grace in black and equation ( 7 ) superposed in red . november 2003 geomagnetic storm periods , particularly where the grace data indicate that t c should be negative . ( a ) values of the t c parameter that were derived from the neutral density measurements on the grace satellite are shown in black , the same values as shown in figure 2b . the red line shows these values that are derived from the global heating , using equations ( 7 ) , ( 8) , ( 9 ) . ( b ) the blue line shows the cooling rate of the thermosphere that is proportional to changes in global no levels , times the temperature , from ( 9 ) . ( c ) like figure 2d , the green line shows the saber no power measurements , while the superposed blue line shows the linear fit of the same values shown in figure 3b , using ( 10 ) . figure 3b shows the value of c no with the blue line , at the 4 min cadence . the levels are different from figure 2 since the values that are plotted are from ( 9 ) rather than ( 3 ) . this quantity indicates the rate of change in temperature per time step , due to the no cooling , in units of k / min . the line in figure figure 3c shows the comparison with the daily , no emissions measured on saber ( green line ) . the blue line shows the result of a linear fit , after smoothing figure 3b to the same 24 h intervals used for the saber measurements : ( 10)nosaber=92 + 2370cno the correlation has increased to 0.92 , even though no saber measurements were used in the fitting process . as the revised fit resulted in a smaller time constant a close comparison of the graphs indicates that the faster decay has a better match with the saber measurements . the mean error is lower as well , at 34 gw , and the standard deviation is 43 gw . while the values shown in figure 3b seem small , a value of 0.6 k / min amounts to a temperature change of 430k if sustained over a 12 h period . figure 4 shows a similar comparison between the derived value of c no and the measured no emissions , over a 4 year period . in figure 4a the values of c no are shown with the 24 h averaging already calculated so that the heights of the peak values are less than in figure 3 . the plot in figure 4b again shows the saber measurements of no emissions ( green line ) and the linear fit with the data from figure 4a ( blue line ) . over this 4 year period the formula for best fit is ( 11)nosaber=42 + 2790cno results from the revised thermosphere temperature calculation described here for the years 2003 through 2006 . ( a ) the blue line shows the cooling rate of the thermosphere due to the effect of no . these are the same as in figure 3c , except the longer time period is shown and the 24 h smoothing has already been applied . ( b ) the green and blue lines are the same as in figure 3c , using the fit in equation ( 11 ) for this longer time interval . the correlations between the two values is still 0.92 for this entire , 4 year period , and the mean error is 30 gw , with a standard deviation of 41 gw . both the declining phase of the solar cycle as well as the periodicity of the 27 day solar rotation are evident in figure 4 . the previous section has compared the measured nitric oxide emissions with variables that are derived from the global heating rates . it is useful to make another comparison directly with the thermosphere temperature rather than the no parameter derived from equations ( 3 ) or ( 9 ) . figure 5 shows one such comparison for the years 2003 through 2006 . in the upper graph figure 5a the thicker , darkred line shows the value of t c calculated from solar indices with ( 1 ) , as used in the jb2008 model . the thinner , red line shows this value with the addition of the t c values , from ( 7 ) , giving the total temperature . the maximum value of this temperature in each day is used here . as in the earlier graphs , figure 5b shows the saber measurements with a green line , and a superposed line shows a linear fit of the total temperature : ( 12)nosaber=659+.984(tc+tc ) comparison of the no emissions measured with saber and the temperature of the thermosphere . ( a ) the thicker , dark red line shows the t c parameter used in the jb2008 model , as calculated with ( 1 ) . the lighter red line shows the sum of this t c plus the t c values that were calculated with ( 7 ) . ( b ) green line shows the saber measurements of no emissions , and the purple line shows the linear fit of the total temperature , from ( 12 ) . for clarity the correlation between the measured no emission and the total temperature is 0.87 , the mean error is 35 gw , and the standard deviation is 51 gw . the mean value of the temperature in each day had been tried for comparison , rather than the maximum value , and this resulted in lower correlation with the no emissions . while this comparison of the no emissions with the total t c has a good correlation , it led to a question about whether an agreement could be found with a more simple calculation from the solar indices . the formula in ( 1 ) requires calculation of an 81 day centered , boxcar average of these indices , which is not possible in real time . using a multiple linear regression with indices having various smoothing lengths and delays , it was found that the best correlation with the no emissions is found with ( 13)nosaber=217 + 1.46tc+0.831f10 + 2.33s100.634m100.221y10 the correlation coefficient is 0.904 , the mean error is 30.7 gw , and the standard deviation is 43.9 gw . as before , the maximum value of t c in each 24 h is used for the comparison with the 24 h nosaber values . this correlation was achieved using a 3 day centered , boxcar that is time shifted by 1 day , for all solar indices . in other words , the solar indices are their mean value from the current day and prior 2 days . if only the f 10 and s 10 indices are used in the regression , then there is only a minuscule reduction in the correlation , to 0.903 , with the same smoothing and delay . the revised formula is ( 14)nosaber=217 + 1.47tc+0.656f10 + 1.62s10 there is only a small change in the mean error , to 30.8 gw , with a standard deviation of 44.1 gw . in other words , the changes are insignificant ; the reason being that these indices tend to be strongly correlated with each other . figure 6 shows the comparison of the solar indices with the saber measurements , using ( 14 ) to generate the purple line in figure 6c . figure 6a shows the f 10 and s 10 indices , drawn with the blue and black lines , with f 10 offset by + 50 solar flux unit ( sfu ) for better clarity . comparison of the no emissions measured with saber with solar indices f 10 and s 10 and the temperature of the thermosphere . ( a ) blue line shows the f 10 index , offset by 50 sfu , and the black line shows the s 10 index . ( b ) the red line shows the t c values that were calculated with ( 7 ) . ( c ) as in the other graphs , the green line shows the saber measurements of no emissions , and the purple line shows the multiple linear regression fit of the other three values from ( 13 ) . a method had been developed to derive predictions of the thermospheric temperature , using the total auroral heating in the polar regions , as summarized in equations ( 2 ) , ( 3 ) , ( 4 ) . a dimensionless variable had been included in order to account for variations in the rate of cooling in the thermosphere , as observed with the neutral density measurements from champ and grace . the more rapid cooling that followed increased levels of heating was attributed to nitric oxide production . this link was not proven at the time , and this followup comparison with the measurements from the saber instrument , as illustrated in figure 2 , shows a very good correlation , 0.88 , for 2003 . in this followup work , in addition to showing convincing evidence that nitric oxide is indeed responsible for enhanced thermosphere cooling , an improved formula is shown . the main shortcoming of the earlier method was that the corrections to the temperature t c in the jb2008 model could not go below zero , while observations indicated that the actual temperature could go below the baseline level , following extreme geomagnetic activity . figure 3 shows that this new temperature correction calculation did go below zero following the 2003 halloween storm , resulting in a better agreement with the neutral density measurements . the variable that represents no in the new formula has a more direct relationship with the power level of the cooling , no emissions , and this power has a higher correlation with the measured emissions , 0.92 , for 2003 . for a 4 year period the correlation is also 0.92 , as illustrated in figure 4 . this level of correlation is quite amazing , in consideration that one quantity is from the optical measurements of no emissions , and the other is derived entirely independently : solar wind and imf measurements were passed through an empirical model and processed with simple differential equations that use scaling factors and time constants that were derived by fitting neutral density measurements . as mentioned in the previous paper , the thermosphere behaves like a calorimeter , such that for a specified change in temperature the amount of energy involved can be estimated . the calculations by burke with the jacchia 1977 model indicated that raising t c or tmin by 1k increases the total energy in the thermosphere above 100 km altitude by 1.0110 j. therefore , a specified change in the global thermosphere temperature is related to the total amount of thermal and potential energy within the system . it was stated that using = 7.0810 k/(gw min ) , or , 1.210 k / gj , in ( 7 ) produced the best agreement with the neutral density measurements from grace . this number indicates that a heating level of 1410 gw in the model is needed to raise t c temperature by 1k in 1 min , increasing the total energy by 1.0110 j. a heat dissipation of 1410 gw over 1 min produces 8.4710 j , which is about 80% of the burke calculation . the auroral particle precipitation can add another 20% or more to what the w05 model calculates [ wilson et al . another way to apply the results by burke is that 1 gw of heating or cooling applied over a 1 min time period should increase or decrease t c by 5.9510 k. referring to the fit results in ( 11 ) , each gw of no radiated power measured by saber is proportional to 2790 times c no , in units of k / min . this results in the no radiation measured by saber cooling the thermosphere at 3.5810 k / min for each gw of radiated power or 61% of the theoretical value . figure 5 shows that the no emissions have a good correlation ( 0.87 ) with the total thermospheric temperature , using the value of t c derived from solar indices from ( 1 ) plus the t c calculated with ( 7 ) . the maximum value of the temperature in each day had a better correlation than the mean temperature value , which is easily explained by the fact that both the peak value and no emissions are affected by the total heating during the previous several hours . figure 5 is also useful to illustrate how the no emissions are influenced by the 27 day periodicity in solar radiation as well as the variation in this radiation over the solar cycle . as mentioned in section 1 , in the publication by lu et al . they had discussed the correlations between the saber no measurements and the f 10.7 index and the flux from the solar euv experiment on the timed satellite . in the separate comparisons , power law functions of these quantities had correlations of 0.85 and 0.82 , respectively . the largest correlation values were found when the indices were lagged by 1 day . time periods with geomagnetic storm enhancements were excluded . to account for storm time heating , a timeaveraged value of the k p index with an exponent of 3.1 was included in their final prediction formulas , and correlations of 0.89 and 0.90 were achieved for a 7 year period . all versions of the mass spectrometer incoherent scatter class thermosphere models [ hedin , 1983 ] also use a 1 day lag for the f 10.7 index . these prior results were one motivation for trying the correlations shown in figure 6 , as well as desire to find a more simple alternative to ( 1 ) to predict thermosphere temperatures and to avoid the use of an 81 day boxcar average . the results illustrated in figure 6 showed that a 0.90 correlation with the saber measurements could be achieved using a linear combination of the t c parameter plus the f 10 and s 10 indices described by tobiska et al . . this correlation was obtained by use of the mean value of these solar indices over 3 days , using the current and prior 2 days . a longstanding problem has been the calculation of neutral density changes in the thermosphere following large geomagnetic storms , as well as the slower variations throughout the solar cycle . this paper describes an improvement on a technique that calculates changes in the global , minimum temperature of the thermosphere , based on solar wind / imf values that are passed through an empirical model of poynting flux flowing into the polar ionospheres . this temperature can be used in the jb2008 model , or future variations , in order to derive the global neutral density values in geospace . a key parameter in this prediction is the total amount of nitric oxide in the thermosphere . enhanced levels of heating and particle precipitation in the ionosphere effectively increase both the temperature and the concentration of no in the thermosphere , which in turn accelerates the rate at which the thermosphere cools . relative variations in no content are used in the temperature calculation , using coefficients that were obtained by a repetitive , least error fit to thermosphere temperatures that were derived from neutral density measurements on the grace satellite . later comparisons with the global rate of no emissions measured with the saber instrument on the timed satellite indicated that there is a very good agreement between the predicted and measured values . the no emissions correlate highly with the total auroral heating that has been integrated over time , and they also correlate well with the temperature of the thermosphere .

abstractobtaining accurate predictions of the neutral density in the thermosphere has been a longstanding problem . during geomagnetic storms the auroral heating in the polar ionospheres quickly raises the temperature of the thermosphere , resulting in higher neutral densities that exert a greater drag force on objects in low earth orbit . rapid increases and decreases in the temperature and density may occur within a couple days . a key parameter in the thermosphere is the total amount of nitric oxide ( no ) . the production of no is accelerated by the auroral heating , and since no is an efficient radiator of thermal energy , higher concentrations of this molecule accelerate the rate at which the thermosphere cools . this paper describes an improved technique that calculates changes in the global temperature of the thermosphere . starting from an empirical model of the poynting flux into the ionosphere , a set of differential equations derives the minimum , global value of the exospheric temperature , which can be used in a neutral density model to calculate the global values . the relative variations in no content are used to obtain more accurate cooling rates . comparisons with the global rate of no emissions that are measured with the sounding of the atmosphere using broadband emission radiometry instrument show that there is very good agreement with the predicted values . the no emissions correlate highly with the total auroral heating that has been integrated over time . we also show that the no emissions are highly correlated with thermospheric temperature , as well as indices of solar extreme ultraviolet radiation .

Introduction Nitric Oxide Emissions Measured With SABER Prior Results From an Empirical Model Improving the Thermosphere Temperature Prediction Nitric Oxide Emissions Compared With Thermosphere Temperatures Discussion Summary and Conclusion

PMC3402101

while males constitute the majority , female adolescent offenders are a sizeable minority of the overall delinquent population . further , those females who become involved in delinquent activities appear to be doing so at a younger age , and they are involved in a wide range of criminal activities , including violent offenses . the goal of this article is to consolidate an empirical base for our current knowledge about female juvenile offenders trauma - related mental health and rehabilitation issues . we searched for studies using pilots , psyclit , psycinfo , and ebscohost electronic databases . accordingly , we present a review of findings from 33 recent studies showing consistently high rates of trauma exposure , ptsd , and common comorbidities among female adolescent offenders . we also examined recent literature on risk and protective factors for female delinquency , as well as treatments for offenders , and found that there was some early representation of trauma and ptsd as important variables to be considered in etiology and treatment . future plans for addressing the mental health needs of female offenders should be better informed by these recent findings about widespread trauma exposure and related psychological consequences . in the past 10 years an impressive number ( 33 ) of new studies on female offenders trauma exposure and related mental health issues have emerged . further , nearly one - fourth of these new studies are from non - us sources . key findings from our review of these studies indicate that severe exposure from multiple types of trauma is most often found among these young women , and that ptsd rates generally exceed 30% . high prevalence rates for other comorbidities , such as depression , substance abuse , anxiety , and suicidality are also reported . given the extensiveness of these findings , it seems clear that severe trauma exposure and serious mental health sequelae are to be expected in high proportions of incarcerated female offenders , both in international and us forensic settings . while there is modest representation of trauma exposure and related psychological disorders among studies of etiology for female delinquency , present treatments for offenders do not specifically target trauma effects . we hope that the results of this review will encourage the inclusion of trauma as a prominent consideration in future treatment planning for female offenders . there is no conflict of interest in the present study for any of the authors .

backgroundwhile males constitute the majority , female adolescent offenders are a sizeable minority of the overall delinquent population . further , those females who become involved in delinquent activities appear to be doing so at a younger age , and they are involved in a wide range of criminal activities , including violent offenses.objectivethe goal of this article is to consolidate an empirical base for our current knowledge about female juvenile offenders trauma - related mental health and rehabilitation issues.methodwe searched for studies using pilots , psyclit , psycinfo , and ebscohost electronic databases.resultsaccordingly , we present a review of findings from 33 recent studies showing consistently high rates of trauma exposure , ptsd , and common comorbidities among female adolescent offenders . we also examined recent literature on risk and protective factors for female delinquency , as well as treatments for offenders , and found that there was some early representation of trauma and ptsd as important variables to be considered in etiology and treatment.conclusionfuture plans for addressing the mental health needs of female offenders should be better informed by these recent findings about widespread trauma exposure and related psychological consequences .

Background Objective Method Results Conclusion Summary Conflict of interest and funding

PMC3713835

statewide surveillance for all invasive h. influenzae disease in alaska has been conducted since 1980 by the centers for disease control s arctic investigations program ( aip ) based in anchorage , alaska . clinical laboratories are requested to send h. influenzae isolates recovered from a normally sterile site ( e.g. , blood , cerebrospinal fluid , pleural fluid , etc . ) in a resident of alaska to aip for confirmation of identity and serotyping . h. influenzae is confirmed by using gram stain and factor x and v requirements ( differentiation disks ; difco laboratories , detroit , mi , usa ) . since 2005 , a small number of h. influenzae cases have also been identified by pcr on specimens from patients whose conditions had been treated with antimicrobial drugs before samples were collected and whose samples from sterile sites were culture negative . pcr amplification of serotype - specific genes was done by using primers and probes as reported by maaroufi , et al . multilocus sequence typing ( mlst ) of 7 housekeeping gene loci was done on all invasive hia isolates according to a previously described method ( 11 ) . the sequence type ( st ) assignments were made by using the h. influenzae mlst website ( http://haemophilus.mlst.net/ ) . clonal complexes were assigned by using the eburst algorithm with software available at the mlst website ( http://www.mlst.net ) . the is1016-bexa deletion was amplified from genomic dna by pcr by using sense is1016 ( 5-attagcaagtatgctagtctat-3 ) and antisense bexa ( 5-caatgattcgcgtaaataatgt-3)primers ( 12 ) . aip uses a standardized form to collect demographic and clinical data from medical records for each reported case . each year , a list of submitted samples is reconciled with culture results from each participating laboratory , and case lists are compared with state - reportable disease data . serotyping of invasive h. influenzae isolates was introduced gradually ; the practice grew from serotyping isolates from 8% of collected samples in 1980 to serotyping 80% by 1983 . because serotyping was intermittent from 1980 to 1982 a case of invasive h. influenzae disease was defined by isolation of h. influenzae from a normally sterile site , including blood , cerebrospinal fluid , pleural fluid , peritoneal fluid , or joint fluid from a resident of alaska . cases in patients with clinical epiglottitis ( who had h. influenzae isolated from an epiglottis swab sample ) were also reportable . the clinical description of h. influenzae infection was determined by a review of the discharge diagnoses in each case - patient s medical record . when case - patients had multiple discharge diagnoses , the diagnoses related to invasive hia infection were ranked according to severity , from highest to lowest , in the following order : meningitis , epiglottitis , pneumonia , pericarditis , osteomyelitis , septic arthritis , and septicemia with unknown focus . we defined an outbreak of invasive hia disease as the occurrence of confirmed cases of invasive hia infection by isolates within the same clonal complex among persons residing in the same region with incidence rates above baseline . population denominator data for alaska were obtained from the alaska department of labor and workforce development website ( http://www.labor.state.ak.us ) . estimates for calculating rates of hia disease in alaska reflect population figures derived from the 2000 and 2010 census counts ; rates were calculated for the years 20022011 . during 20022011 , alaska s population ranged from 640,841 to 722,190 ; alaska native peoples comprised 19% of the population . seventy - five percent of the population resided in urban centers ; the remaining population was dispersed widely across 586,412 square miles . we defined region a as the southwestern region of alaska , which has a population of 25,000 persons . we identified 958 cases of invasive h. influenzae disease during 19832011 ( data not shown ) ; of these , 858 isolates were available for serotyping by slide agglutination . eight additional culture - negative sterile site samples obtained from patients with clinically compatible illnesses who had been treated with antimicrobial drugs were identified , and isolates were serotyped by using pcr . among samples serotyped , 617 were serotype b and 158 were nontypeable . of the remaining 91 typeable , non serotype b isolates , 44 ( 48% ) were serotype f , 32 ( 35% ) were serotype a , 13 ( 14% ) were serotype e , and 2 ( 2% ) were serotype d ( table 1 ) . hif and hie isolates were identified from 1985 onward ; the first hia isolate was identified in 2002 . during 19832001 , none of the 30 encapsulated , non serotype b isolates were hia ; however , from 2002 through 2011 , 32 ( 52% ) of 62 non serotype b isolates were hia ( p<0.001 vs. 19832001 ) ( figure 1 ) . haemophilus influenza type b conjugate vaccine introduction in alaska . reported cases of non - b encapsulated haemophilus influenza disease , alaska , 19832011 . among the 32 hia cases , 28 ( 88% ) occurred in children < 2 years of age , 1 ( 3% ) occurred in a child 24 years , and 3 ( 9% ) occurred in persons > 5 years ( range 848 years ) . the median age of case - patients was 0.7 years ( range 0.348 years ) . ethnicity data were available for all cases ; 28 ( 88% ) occurred in alaska native persons . cases occurred in 5 areas of alaska ; 21 ( 66% ) cases occurred in region a ( figure 2 ) . among all invasive hia cases , the most common clinical syndromes were meningitis ( 12 [ 38% ] ) , pneumonia with bacteremia ( 11 [ 34% ] ) , and septic arthritis ( 6 [ 19% ] ) . all cases of meningitis occurred in children < 2 years of age ; there were no cases of epiglottitis . three case - patients < 1 year of age died ( case - fatality ratio 9% ) . twenty - five ( 78% ) case - patients < 5 years of age had been vaccinated for hib at appropriate ages . two distinct outbreaks , 1 in 2003 ( 5 cases ) ( 9 ) and the other in 20092011 ( 15 cases ) , comprised 63% of the 32 invasive hia cases that occurred from 20022011 . although there were 20 hif cases during the same period , the epidemiology differs from hia cases ; hif cases tend to occur in older persons ( median age of case - patients : 55 years ) , in non alaska native persons ( 70% ) , and a higher proportion of case - patients ( 45% ) have a clinical syndrome of pneumonia with bacteremia . geographic distribution of invasive haemophilus influenzae type a disease in alaska , by sequence type ( st ) . a ) st 576 ; b ) st 23 ; c ) st 56 . from december 1 , 2009 , through december 31 , 2011 , 15 cases of hia occurred in region a , 10 ( 67% ) of which were identified either in the largest community ( 4 cases ) or in nearby villages ( 6 cases ) . thirteen of the 15 cases occurred in alaska native children < 2 years of age ; 1 was in an alaska native child 8 years of age , and 1 was in a non - native adult , who was 48 years of age . the median age of case - patients was 0.8 years ( range 0.348 years ) . of the 13 cases in alaska native children < 5 years of age , 8 ( 62% ) were in girls . primary clinical conditions included meningitis ( n = 6 ) , pneumonia with bacteremia ( n = 3 ) , septic arthritis ( n = 2 ) , cellulitis ( n = 1 ) , and bacteremia ( n = 1 ) . four case - patients had underlying conditions , including a history of otitis media , chronic pulmonary disease , premature birth , esophageal reflux , abnormal heart murmur , or pulmonary valve stenosis , and 1 child experienced a stroke while hospitalized . of these children , 1 was not vaccinated with prp - omp at the appropriate age . the overall rate of invasive hia disease in alaska during 20022011 was 5.4/100,000 population ( 95% ci 3.67.7 ) among children < 5 years of age ; however , the rate in alaska native children < 5 years was 36 times higher than in non - native children ( 18/100,000 versus 0.5/100,000 , p < 0.0001 ) . overall rates of invasive hib and hif disease in children in alaska < 5 years of age during 20022011 were 2.8/100,000 ( 95% ci : 1.64.6 ) and 0.9/100,000 ( 95% ci : 0.32.2 ) , respectively . invasive hia disease occurred primarily in region a , where the rate among children < 5 years of age was 72.3/100,000 compared with 1.2/100,000 in children < 5 years of age in the rest of the state ( p<0.0001 ) . during 2010 , the year in which the most cases occurred during the most recent outbreak , the rate of invasive hia disease in children < 5 years of age in region a was 250/100,000 compared to the rate for previous years of 45/100,000 ( p<0.001 fisher exact test ) . mlst of the 27 hia isolates yielded 3 sequence types ( sts ) , which are shown by month and year ( table 2 ; figure 2 ) . eburst analysis showed that these 3 sts fell into a single clonal complex : cc23 . st576 was identified 4 times during the 26-year surveillance period and is a double locus variant of st23 . all of the st56 isolates , which are single locus variants of st23 , were found during 20082011 . each of these sts first appeared in a port or major city in alaska and was subsequently isolated from samples from patients in the western rural regions of the state ( figure 2 ) . we identified 958 cases of invasive h. influenzae disease during 19832011 ( data not shown ) ; of these , 858 isolates were available for serotyping by slide agglutination . eight additional culture - negative sterile site samples obtained from patients with clinically compatible illnesses who had been treated with antimicrobial drugs were identified , and isolates were serotyped by using pcr . among samples serotyped , 617 were serotype b and 158 were nontypeable . of the remaining 91 typeable , non serotype b isolates , 44 ( 48% ) were serotype f , 32 ( 35% ) were serotype a , 13 ( 14% ) were serotype e , and 2 ( 2% ) were serotype d ( table 1 ) . hif and hie isolates were identified from 1985 onward ; the first hia isolate was identified in 2002 . during 19832001 , none of the 30 encapsulated , non serotype b isolates were hia ; however , from 2002 through 2011 , 32 ( 52% ) of 62 non serotype b isolates were hia ( p<0.001 vs. 19832001 ) ( figure 1 ) . haemophilus influenza type b conjugate vaccine introduction in alaska . reported cases of non - b encapsulated haemophilus influenza disease , alaska , 19832011 . among the 32 hia cases , 28 ( 88% ) occurred in children < 2 years of age , 1 ( 3% ) occurred in a child 24 years , and 3 ( 9% ) occurred in persons > 5 years ( range 848 years ) . the median age of case - patients was 0.7 years ( range 0.348 years ) . ethnicity data were available for all cases ; 28 ( 88% ) occurred in alaska native persons . cases occurred in 5 areas of alaska ; 21 ( 66% ) cases occurred in region a ( figure 2 ) . among all invasive hia cases , the most common clinical syndromes were meningitis ( 12 [ 38% ] ) , pneumonia with bacteremia ( 11 [ 34% ] ) , and septic arthritis ( 6 [ 19% ] ) . all cases of meningitis occurred in children < 2 years of age ; there were no cases of epiglottitis . three case - patients < 1 year of age died ( case - fatality ratio 9% ) . twenty - five ( 78% ) case - patients < 5 years of age had been vaccinated for hib at appropriate ages . two distinct outbreaks , 1 in 2003 ( 5 cases ) ( 9 ) and the other in 20092011 ( 15 cases ) , comprised 63% of the 32 invasive hia cases that occurred from 20022011 . although there were 20 hif cases during the same period , the epidemiology differs from hia cases ; hif cases tend to occur in older persons ( median age of case - patients : 55 years ) , in non alaska native persons ( 70% ) , and a higher proportion of case - patients ( 45% ) have a clinical syndrome of pneumonia with bacteremia . geographic distribution of invasive haemophilus influenzae type a disease in alaska , by sequence type ( st ) . from december 1 , 2009 , through december 31 , 2011 , 15 cases of hia occurred in region a , 10 ( 67% ) of which were identified either in the largest community ( 4 cases ) or in nearby villages ( 6 cases ) . thirteen of the 15 cases occurred in alaska native children < 2 years of age ; 1 was in an alaska native child 8 years of age , and 1 was in a non - native adult , who was 48 years of age . the median age of case - patients was 0.8 years ( range 0.348 years ) . of the 13 cases in alaska native children < 5 years of age , 8 ( 62% ) were in girls . primary clinical conditions included meningitis ( n = 6 ) , pneumonia with bacteremia ( n = 3 ) , septic arthritis ( n = 2 ) , cellulitis ( n = 1 ) , and bacteremia ( n = 1 ) . four case - patients had underlying conditions , including a history of otitis media , chronic pulmonary disease , premature birth , esophageal reflux , abnormal heart murmur , or pulmonary valve stenosis , and 1 child experienced a stroke while hospitalized . of these children , 1 was not vaccinated with prp - omp at the appropriate age . the overall rate of invasive hia disease in alaska during 20022011 was 5.4/100,000 population ( 95% ci 3.67.7 ) among children < 5 years of age ; however , the rate in alaska native children < 5 years was 36 times higher than in non - native children ( 18/100,000 versus 0.5/100,000 , p < 0.0001 ) . overall rates of invasive hib and hif disease in children in alaska < 5 years of age during 20022011 were 2.8/100,000 ( 95% ci : 1.64.6 ) and 0.9/100,000 ( 95% ci : 0.32.2 ) , respectively . invasive hia disease occurred primarily in region a , where the rate among children < 5 years of age was 72.3/100,000 compared with 1.2/100,000 in children < 5 years of age in the rest of the state ( p<0.0001 ) . during 2010 , the year in which the most cases occurred during the most recent outbreak , the rate of invasive hia disease in children < 5 years of age in region a was 250/100,000 compared to the rate for previous years of 45/100,000 ( p<0.001 fisher exact test ) . mlst of the 27 hia isolates yielded 3 sequence types ( sts ) , which are shown by month and year ( table 2 ; figure 2 ) . eburst analysis showed that these 3 sts fell into a single clonal complex : cc23 . st576 was identified 4 times during the 26-year surveillance period and is a double locus variant of st23 . all of the st56 isolates , which are single locus variants of st23 , were found during 20082011 . each of these sts first appeared in a port or major city in alaska and was subsequently isolated from samples from patients in the western rural regions of the state ( figure 2 ) . the 20092011 outbreak investigation of invasive hia disease in region a , highlighted the emergence of a pathogen that was first identified in alaska in 2002 . three mlst sts were identified during the 10-year period of 20022011 ; the same 3 sts were recently identified in british columbia by shuel , et al . although the first appearance of each st occurred in a port or major city outside of region a , outbreaks of disease occurred only in region a , the region that reported the highest rate of childhood respiratory hospitalizations in the state and the highest rate of hib disease in the prevaccine era ( 3 ) . patterns of spread of these hia sts showed a tendency of slow expansion within a limited geographic region over a period of years . extensive evaluation of isolates of invasive hi disease ( 19832011 ) , and a large study of h. influenzae throat carriage in 1982 ( 14 ) revealed no confirmed isolates of hia in alaska before 2002 . reports of hia carriage in children in alaska during the early 1980s ( 14 ) and invasive hia disease before 2002 ( 3 ) were incorrect ; subsequent mlst evaluation of these isolates and confirmatory re - evaluation by serotyping determined that they were either nontypeable h. influenzae or encapsulated h. influenzae of other serotypes . suboptimal accuracy of serotyping by slide agglutination alone for identification of capsular types in general ( 15 ) and hia in particular ( 16 ) has recently been well characterized and likely explains the original false - positive hia results . hia was not identified in alaska until 2002 and has since increased and caused outbreaks . although it is possible that hia existed in alaska before 2002 , it is unlikely that it was commonly circulating during the 1980s and 1990s . the primary focus of the surveillance and carriage studies in those decades was hib , and thus , surveillance may have missed an occasional hia case . serotyping for non type b encapsulated strains was routinely performed on available h. influenzae isolates in alaska from 1985 onward , and multiple isolates of nontypeable h. influenzae , hid , hie , and hif were identified over the 32 years covered in this report . the subsequent rapid expansion throughout the rest of the study period also suggests recent introduction , as opposed to the uncovering of a previously prevalent strain , as apparently occurred for hia disease in the american indian population in the american southwest after introduction of the hib conjugate vaccine ( 17 ) . single episodes of invasive disease caused by each of the 3 hia sts were initially identified outside of region a ; later appeared within region a , where they proliferated and spread . although disease outside of region a remained rare , within region a , invasive hia disease caused by 2 of the 3 st groups expanded slowly , but persistently , to contiguous geographic areas . in the central part of region a , rates of hia disease during the 20092011 outbreak among children < 5 years of age approached those of prevaccine invasive hib disease in alaska ( 250/100,000 ) ( 3 ) . the disparity in rates of disease by geographic region may be partly explained by several factors that are known to contribute to the elevated risk for respiratory infection in general in region a. previous studies have shown associations between the rate of respiratory infections and household crowding , decreased availability of in - home piped water service , indoor smoke , and poverty ( 18,19 ) . each of these factors could play a role in facilitating transmission or invasiveness of hia disease in human populations . these same factors likely play a role in other populations for whom high rates of hia disease are routinely described , including residents of the american southwest ( 17 ) and northwestern ontario , canada ( 5 , 6 ) , although the factors contributing to increased rates of disease in utah are less clear ( 7 ) . recently , invasive hia cases have also been identified in british columbia and manitoba , canada ( 13 , 20 ) . in contrast , hia is not a major cause of invasive h. influenzae disease in other areas of canada , the united states , and europe , where these risk factors are not as prevalent and hif plays a more prominent role ( 2123 ) . it should also be noted that in the other areas of alaska , where the above - mentioned contributory factors are less prevalent , the rate of invasive hia disease between 2002 and 2011 ( 0.08/100,000 ) was similar to previously noted rates in the rest of the united states before vaccine introduction ( 0.04/100,000 ) ( 24 ) . the clinical syndrome associated with hia infection in alaska from 20022011 was similar to that described in canada , utah , and the american southwest ( 5,7,17,20 ) : most cases occurred in children < 2 years of age , 84% of case - patients were hospitalized , and the case - fatality ratio was 9% . of the 13 alaska native children infected during the 20092011 outbreak , 6 ( 46% ) had meningitis , 1 ( 8% ) child died , and 1 child had serious neurologic complications following a stroke the child experienced in the hospital . although clinical disease associated with hia was often severe , the hia isolates from this outbreak in alaska do not exhibit the is1016-bexa partial deletion , a characteristic classically associated with more severe disease ( 12,25 ) . the severity of disease and increasing rates in recent years have increased interest in identifying prevention and control options . a promising hia conjugate vaccine , modeled after the hib conjugate vaccines , has been suggested for control of invasive hia disease ( 4 ) . however , the population at risk for high rates of hia disease is so small that further development of this potentially useful tool has not progressed . the presumptive ecologic and transmission characteristics of hia disease , which rely heavily on analogies with invasive hib disease , raise the possibility that interrupting person - to - person transmission through chemoprophylaxis may be effective . current clinical practice in region a often includes chemoprophylaxis of close and household contacts of hia case - patients ( 26 ) . however , to this point , there are no reports of secondary invasive disease cases ( i.e. , a case of invasive hia disease occurring in a close or household contact of a case - patient with hia disease ) , and thus , a key component of the justification for a chemoprophylaxis control strategy is missing : identification of those at risk for secondary disease once a sentinel case has occurred . finally , several identified risk factors for increased respiratory disease are modifiable , and continuing efforts are underway to address issues of lack of running water , housing ventilation , indoor wood smoke , and other factors , in the high risk area , which may lead to reduced rates of hia disease . to better define carriage and transmission patterns and further clarify risk factors for invasive hia disease , aip is currently evaluating risk factors for disease and hia carriage among contacts and noncontacts of case - patients in alaska . these data may contribute to future recommendations for prevention and control of hia disease in this environment . the primary limitations of this study are a result of the changing laboratory and surveillance methodologies used over the past 3 decades . although statewide surveillance of invasive h. influenzae disease began in 1980 , it was primarily focused on hib disease , and compliance with the program expanded gradually . we can not , therefore , confirm that all invasive h. influenzae isolates from the early years of surveillance were sent to aip , or that there was not preferential selection of hib strains for surveillance . however , by 1983 , both nontypeable and non - b encapsulated strains were being received and identified routinely in the aip laboratory . these isolates were retained , and all hia strains were subsequently evaluated by mlst by aip laboratory personnel to confirm strain identity . although earlier serotyping results suggested the presence of invasive hia disease in the mid-1990s ( and hia in carriage studies from the early 1980s ) , subsequent molecular testing and re - serotyping resulted in reclassification of all of these isolates , mostly to nontypeable h. influenzae . this experience illustrates the importance of molecular methods in evaluation of epidemiologic patterns , inter - laboratory quality control ( 27 ) , and the importance of close collaboration between laboratory and epidemiology groups . we confirmed the presence of hia in alaska in 2002 , and its subsequent emergence as a cause of invasive bacterial disease in an area in rural alaska associated with high risk for the disease . its pattern of spread is consistent with slow , person - to - person transmission , with persistence in the high - risk area . studies currently in progress in alaska and other populations should help shape those efforts by providing additional information on risk factors and disease transmission .

before introduction of haemophilus influenzae type b ( hib ) vaccines , rates of hib disease in alaska s indigenous people were among the highest in the world . vaccination reduced rates dramatically ; however , invasive h. influenzae type a ( hia ) disease has emerged . cases of invasive disease were identified through alaska statewide surveillance during19832011 . of 866 isolates analyzed for serotype , 32 ( 4% ) were hia . no hia disease was identified before 2002 ; 32 cases occurred during 20022011 ( p<0.001 ) . median age of case - patients was 0.7 years ; 3 infants died . incidence of hia infection ( 20022011 ) among children < 5 years was 5.4/100,000 ; 27 cases occurred in alaska native children ( 18/100,000 ) versus 2 cases in non - native children ( 0.5/100,000 ) ( risk ratio = 36 , p<0.001 ) . from 12/2009 to 12/2011 , 15 cases of hia disease occurred in southwestern alaska ( in children < 5 years , rate = 204/100,000 ) . since introduction of the hib conjugate vaccine , hia infection has become a major invasive bacterial disease in alaska native children .

Methods Results Descriptive Epidemiology Invasive Hia Cases, 20022011 Description of the 20092011 Hia Outbreak Incidence Rates Subtyping Data Discussion

PMC4101197

stroke represents the third leading cause of death in industrialized nations , after myocardial infarction and cancer , and the single most common reason for permanent disability . a new optimism emerged after the advent of intravenous thrombolysis with recombinant tissue plasminogen activator ( rtpa , alteplase ) and , since then , stroke treatment has dramatically changed . intravenous ( iv ) thrombolysis with tissue plasminogen activator ( tpa , alteplase ) is the standard of care in the treatment of acute ischemic stroke in current clinical practice and the extension of the time window up to 4.5 hours after symptoms onset has been already approved by the european medicines agency ( emea ) and us food and drug administration ( fda ) agency and included in european and american guidelines recommendations . despite iv tpa for stroke has become widely used in different countries since 1996 , its use has several limitations such as short therapeutic window , low arterial recanalization rate , risk of major bleeding , moderate effect on non - selected patients and several exclusion criteria and contraindications leading to a low proportion of treated patients . all these facts have contributed , over the last decade , to develop and study new thombolytic drugs , new routes of administration , longer treatment windows of treatment and different mechanical devices to locally remove the clot . clinical outcome in ischemic stroke has been shown to be strongly linked to revascularization in numerous independent studies [ 4 - 6 ] . a meta - analysis of more than fifty studies evaluating spontaneous or therapeutic arterial recanalization demonstrated a strong correlation between arterial recanalization and good prognosis . randomized clinical trials with iv [ 7 , 8 ] and intra - arterial tpa have established that good clinical outcome after successful recanalization is time - dependent . however , thrombolytic therapy in longer therapeutic windows has been associated with improved outcomes when reperfusion / recanalization occurs when patient selection is based on mismatch concept using multimodal neuroimaging [ 10 , 11 ] . therefore , clinical and experimental research in acute ischemic stroke is continuously providing new strategies of acute management using pharmacological or interventional endovascular approaches and promoting the use of multimodal neuroimaging techniques as a treatment - selection tool . this article provides a comprehensive review of intravenous thrombolytic treatment and endovascular therapy in acute ischemic stroke based on the largest prospective studies and randomized clinical trials published to date ( table 1 ) . the national institute of neurological disorders and stroke ( ninds ) tissue plasminogen activator clinical trial demonstrated for the first time substantial benefit from the use of iv tpa in patients with acute ischemic stroke within 3 hours of the onset of stroke symptoms . in this study , patients treated with tpa were more likely to have a favorable neurological outcome at 90 days ( or 1.7 ; 95 % ci , 1.2 - 2.6 ; p=0.008 ) . compared to controls , tpa recipients had a 32 % relative increase in the likelihood of minimal or no disability with a 10-fold increase ( 6.4 % vs 0.6 % ) in symptomatic intracerebral hemorrhage ( shic ) . despite four other phase iii clinical trials with tpa showed no positive results and failed in demonstrating the benefit of tpa a pooled analysis of the first 6 iv tpa trials ( ecass 1 , ecass 2 , atlantis a , atlantis b , ninds 1 and ninds 2 ) confirmed the benefit of tpa up to 3 hours and suggested a potential benefit beyond 3 hours for some patients . after its aproval in europe , the safe implementation of thrombolysis in stroke - monitoring study , sits - most , indicated that , overall , routine clinical use of tpa within 3 hours of stroke onset is safe and effective out of randomized clinical trials . in addition , the randomized clinical trial ecass iii showed a modest but clear benefit in terms of favorable outcomes for iv tpa in the 3 to 4.5-hour window as compared to placebo . the results from ecass iii are in consistency with the previously stated time dependency on the effect of iv tpa . in the 3 - 4.5-hour window of ecass iii , fourteen patients were needed to be treated in order to achieve one additional favorable outcome as compared to 8 patients in the 0 - 3.0 hour window from the ninds trial . the sits - international stroke treatment registry ( sits - istr ) is a prospective , multinational , internet - based registry of unselected patients who are given iv tpa thrombolysis for acute stroke in accordance with broadly accepted guidelines . the sits - istr investigators compared the outcomes of patients who received full dose iv rt - pa between 3 - 4.5 hours ( n=664 ) versus within 0 - 3 hours ( n=11,865 ) after ischemic stroke onset in clinical practice . the median time from symptoms onset to treatment was 195 minutes and 140 minutes , respectively . there was no difference in the main outcomes between the two groups even after adjusting for clinical trial prognostic variables . in agreement with the findings from the pooled analysis of the previous iv rt - pa trials , ecass iii and sits - istr did not raise any safety concerns about iv thrombolysis at its later time window regardless of the lack of advanced imaging modalities to exclude presumed high - risk patients . importantly , beyond 4.5 hours from stroke onset , no net therapeutic benefit has been demonstrated and a meta - analysis of clinical trials with alteplase , including data from ecass iii and epithet , suggest an increased risk of mortality ( or 1.49 , 95 % ci , 1.00 - 2.21 ) when alteplase is given in the 4.5 to 6 hours window from symptoms onset . european license of iv tpa treatment has several restrictions on its use which have been adopted from the inclusion and exclusion criteria used in the randomized clinical trials . in many circumstances no evidence - based data are available and recommendations are based on expert judgments . some of these restrictions are not considered by the united states and canadian license and stroke guidelines . of particular relevance because of their frequency are treating patients aged 80 years or older and those with prior stroke and concomitant diabetes . recent data from the prospective registry sits - istr ( safe implementation of thrombolysis in stroke - international stroke thrombolysis registry ) and the virtual archive of international clinical trials ( vista ) showed that , despite a worse outcome in patients > 80 years compared with younger , thrombolytic therapy with iv tpa is associated with better functional outcome in both age groups [ 16 - 18 ] . in the international and multicentre third international stroke trial ( ist-3 ) , 3035 patients within 6 hours of symptoms onset were allocated to 0.9mg / kg intravenous tpa or to control . the study concluded that thrombolysis within 6 hours improves functional outcome and the benefit do not seem to be diminished in elderly patients . so , these results should , therefore , encourage clinicians to consider thrombolytic treatment for patients aged over 80 years . with respect to treating patients who had the combination of previous stroke and diabetes mellitus , although blood glucose levels > 180 mg / dl are associated with poor outcome and shic , functional outcome in alteplase - treated patients is better than in controls among diabetics , patients with prior stroke or with the coexistence of both factors . currently , tpa is the only approved drug that can lead to recanalization of occluded vessels and restore brain circulation before irreversible damage has happened in order to improve clinical outcome of patients treated at any age and in the presence of factors risk such as diabetes and previous stroke . there is growing evidence to support the notion of selecting patients for reperfusion therapy on the basis of brain tissue status as opposed to time from stroke onset . three distinct regions can be identified in the ischemic brain according to the severity of hypoperfusion : ( 1 ) brain that is non - functional and irreversibly damaged ( infarct core ) ; ( 2 ) potentially salvageable hypoperfused brain that is functionally impaired but structurally intact and is destined to undergo infarction in the absence of reperfusion ( penumbra ) ; ( 3 ) hypoperfused brain that is both functionally and structurally intact and will not undergo infarction even in the absence of reperfusion ( benign oligemia ) [ 22 , 23 ] . the hypothesis states that the higher is the mismatch between the infarct core and the penumbral tissue the higher will be the benefit from reperfusion regardless of how much time has elapsed since stroke onset . multimodal magnetic resonance imaging ( mri ) allows , through the diffusion and perfusion sequence , establish those areas of brain tissue in which there is an alteration of cerebral blood flow ( perfusion weithed imaging - pwi ) without irreversible damage to the brain parenchyma ( diffusion weithed imaging - dwi ) . multiple retrospective clinical studies support the use of multimodal mri to select patients eligible for thrombolytic therapy beyond three hours after onset of symptoms [ 24 - 26 ] . an mri perfusion study demonstrated that as many as 70 - 80 % of the patients with proximal arterial occlusion may have a significant mismatch as far as 9 to 24 hours post stroke onset . these studies imply that the therapeutic window may be protracted in selected cases and this constitutes the rationale for multimodal imaging selection . although the role of multimodal ct perfusion study in detection of ischemic tissue at risk is recognized , its value for selecting eligible patients for thrombolysis in longer time windows is controversial . the use of this technique for thrombolysis selection patients was associated with better functional outcomes ( adjusted or 2.88 , 95 % ci , 1.50 - 5.52 ) compared with simple ct scan , mainly in patients treated after 3 hours . two main strategies applying imaging selection to clinical trials have been adopted . in the first one , this approach has been used in the dwi evolution for understanding stroke etiology ( defuse ) and echoplanar imaging thrombolysis evaluation(epithet ) trials and tests the hypothesis that patients with mismatch patterns will respond to treatment while those without mismatch patterns will not . the second approach uses perfusion imaging in order to only select patients with favorable mismatch patterns . this approach has been employed in the desmoteplase in acute stroke ( dias ) and dose escalation of desmoteplase(dedas ) studies [ 31 , 32 ] . the pooled analysis of the two phase ii dose - finding randomized trials of iv desmoteplase within 3 - 9 hours of ischemic stroke onset in patients with dwi / pwi mismatch on mri showed very promising results . the phase iii dias-2 trial , however , did not confirm the benefit of this treatment strategy . the failure of dias-2 was largely attributed to the high response rate in the placebo group that was presumably related to the mild strokes and low proximal middle cerebral artery ( mca ) occlusions rate enrolled in the study . the ongoing dias-3 and dias-4 trials are no longer based on the mismatch concept . in these trials , ischemic stroke patients with proximal arterial occlusion or high - grade stenosis on mri or cta and a baseline nihss score of 4 - 24 are randomized to receive either 90g / kg desmoteplase or placebo within 3 - 9 hours after the onset of stroke symptoms and should definitely elucidate if thrombolysis with iv desmoteplase up to 9 hours is safe and effective ( clinicaltrials.gov identifier : nct00790920 ) . the defuse study was a prospective multicenter study in which 74 consecutive stroke patients were treated with iv rt - pa 3 to 6 hours after symptom onset . early reperfusion was associated with favorable clinical response in patients with a dwi / pwi mismatch ( or , 5.4 ; p=0.039 ) . conversely , patients with no identifiable mismatch did not appear to benefit from early reperfusion . malignant profile defined as a baseline dwi lesion 100ml and/or a pwi lesion 100ml with 8 seconds of tmax delay . the authors concluded that baseline mri could identify patient subgroups that are likely to benefit from reperfusion therapies as well as subgroups that are unlikely to benefit or may be harmed by it . while the aforementioned studies have failed to show a definite benefit for multimodal imaging - based iv thrombolysis at late time windows , they have demonstrated the overall safety of this approach with sich rates equal or lower than what has been seen with non - contrast ct - based iv thrombolysis within the 0 - 3 hour window . although delayed treatment according to mismatch selection can not be widely recommended as part of routine care , new prospective phase iii trials are running ongoing to validate the mismatch selection paradigm . the extra time for thrombolysis in emergency neurological deficits ( extend ) ( clinicaltrials.gov identifier : nct00887328 ) and the european cooperative acute stroke study-4 ( ecass-4 ) trials will select patients with mri mismatch and a 4.5- to 9-hour time window by an automated online estimation of penumbra by the rapid program , whereas the imaging - based thrombolysis trial in acute ischemic stroke - ii ( itais - ii ) is a prospective , blinded , controlled study that aims to study the safety and efficacy of multiparametric ct - based iv thrombolysis within 3 to 9 hours of stroke onset . to test the safety of iv tpa in patients with unknown stroke onset or wake - up stroke , the efficacy and safety of mri - based thrombolysis in wake - up stroke trial ( wake - up ) has already started ( clinicaltrials.gov identifier : nct01525290 ) . this is a randomised , double - blind , placebo - controlled phase iii clinical trial of mri based thrombolysis in acute stroke patients with unknown time of symptoms onset and last time seen well > 4.5 hours or patients with stroke symptoms recognized on awakening with a dwi - flair mismatch pattern indicative of acute ischemic stroke less than 4.5 hours of age . iv administration of tenecteplase dose escalation ( 0.1 , 0.25 , and 0.4 mg / kg ) was compared with the standard dose of tpa 0.9 mg / kg in 112 patients with acute ischemic stroke within 3 hours after symptoms onset . the trial was prematurely stopped due to a lower clinical response and an increased risk of symptomatic hemorrhage in the 0.4 mg / kg dose of tenecteplase . later , an innovative design in a small sample of patients suggests that in patients with a proximal occlusion of the middle cerebral artery refractory to iv tpa treatment , a single bolus of tenecteplase ( 0.1 mg / kg ) is safe and effective . recanalization rate at 24 hours was 100 % and favorable outcome was observed in 69 % out of the 13 patients treated with both thrombolytic agents . recently , the results of a phase 2b , randomized clinical trial to compare the standard dose of alteplase with two different doses of tenecteplase ( 0.1 and 0.25 mg / kg ) in the 6-hour window were published . the higher dose of tenecteplase was superior to the lower dose and to alteplase for all efficacy outcomes . a phase 3 trial of tenecteplase versus alteplase in the time window that is currently approved for thrombolysis would be needed . compared with intravenous therapy , intra - arterial ( ia ) farmacological therapy has the advantage of providing a higher concentration of lytic agent delivered to the clot target while minimizing the systemic exposure to drug and it has also the potential for greater efficacy with higher recanalization rates . this technique allows as well , the use of catheters to directly deliver a clot - disrupting or retrieval device to a thromboembolus that is occluding a cerebral artery . this last approach offers theoretical advantages over pharmacological thrombolysis , such as the rate and speed of recanalization , reduced risk of ich and longer time window for use . however , mechanical approaches are particularly associated with greater technical difficulty , excessive trauma to the vasculature potentially leading to vasospasm , vessel dissection , perforation or rupture , and fragmented thrombus causing distal embolization into previously unaffected territories . moreover , the disadvantages of endovascular treatment over intravenous , in general , include additional time required to initiate therapy and availability only at specialized centres . intra - arterial thrombolysis has been tested only in a few controlled trials including ischemic stroke patients with anterior circulation occlusions . there have been no published randomized controlled trials of endovascular therapy that have included basilar artery occlusions . the prolyse in acute cerebral thromboembolism ii ( proact ii ) study demonstrated the safety and efficacy of ia thrombolysis in patients with an mca occlusion . one hundred and eighty subjects were randomized within 6 hours to treatment with ia pro - urokinase ( uk ) and iv heparin or iv heparin alone . patients in the pro - uk group had a greater recanalization rate ( 66 % versus 18 % ) and a better functional outcome at 3 months ( 40 versus 25 % ) than patients in the control group . although sich rate was greater in the pro - uk group ( 10 % versus 2 % ) , overall mortality rates were similar in the two treatment arms ( 25 % versus 27 % ) . the middle cerebral artery embolism local fibrinolytic intervention trial ( melt ) investigated ia urokinase versus placebo up to 6 hours after stroke onset in patients with a mca occlusion . the study was stopped when iv alteplase was approved in the 0 to 3-hour window but a substantial benefit was observed for the secondary end point of excellent ( mrs 0 - 1 at 3 months ) functional outcome ( 42.1 % versus 22.8 % ) . a meta - analysis of randomized , controlled trials evaluating 395 patients supported the benefit of ia thrombolysis for good ( or , 2.05 ; 95 % ci , 1.33 - 3.14 ) and excellent ( or , 2.14 ; 95 % ci , 1.31 - 3.51 ) outcomes . combined or bridging iv and ia pharmacological thrombolysis is a reperfusion strategy investigated in several non - controlled trials that has the benefit of faster initiation of iv treatment followed by rescue ia revascularization in patients who have not had a successful recanalization after iv treatment . the emergency management of stroke trial ( ems ) and the interventional management of stroke trial ( ims ) demonstrated that the combined iv ( 0.6mg / kg)/ ia ( up to 22 mg ) tpa approach had similar rates of mortality and sich compared with subjects of similar severity and age treated with iv tpa alone in the ninds stroke trial , although no difference was observed in clinical outcome [ 42 - 44 ] . the ims ii study added the ekos micro - infusion catheter to the protocol which uses acoustic streaming to increase fluid penetration , thus driving the thrombolytic agent into the thrombus . the ims ii results showed a higher recanalization rate than in ims i ( 73 % versus 56 % ) , but there were not statistically significant differences with respect to functional outcome at 3 months ( 46 % versus 43 % ) . a case - control study of 42 patients treated with bridging iv / ia tpa rescue therapy compared with 84 historical non - responder patients to iv tpa ( persistent arterial occlusion 1 hour after tpa ) showed that combined thrombolysis increases recanalization rate ( 45.2 % versus 18.1 % at 12 hours ) and the likelihood of good outcome at 3 months ( 40 % versus 14.9 % ) . merci retriever was the first thrombectomy device used in a randomized clinical trial to evaluate the mechanical thrombectomy approach in the treatment of acute stroke . first patients treated as part of the mechanical embolus removal in cerebral ischemia ( merci ) multicenter safety trial were published in 2004 . after that , successive generations of the merci device have been reported in three prospective , single - arm , non - randomized multicenter studies with progressively better safety and outcome results [ 46 - 48 ] . this device is a spring - like device made of nitinol connected to the end of a wire with coil loops that is used with a microcatheter and a balloon - guided catheter ( fig . multimerci trial , using a newer - generation thrombectomy device ( l5 retriever ) , included patients within 8 hours of stroke onset who had either failed to respond to iv tpa or were ineligible for iv tpa . recanalization was achieved in 55 % with the device alone , increasing to 68 % with combined mechanical and ia thrombolytic therapy , and sich occurred in 9.8 % of patients . overall , clinically significant procedural complications occurred in 5.5 % and mortality in 34 % . a favourable outcome ( modified rankin score of 2 ) was seen in 36 % of patients at 90 days . in a pooled analysis of the merci and multi merci studies including 305 patients , successful recanalization was independently associated with good outcome ( or , 20.4 ; 95 % ci , 7.7 to 53.9 ) and reduced mortality ( or , 0.28 ; 95 % ci , 0.16 to 0.50 ) . importantly , the higher was the degree of recanalization the more frequent was favourable outcome , so that for each increase in timi grade , the odds of a good outcome increased 2.6-fold ( 95 % ci ; 1.9 to 3.4 ) . previous iv tpa administration to mechanical embolectomy did not increase the risk of sich ( 10.4 % versus 8.6 % ) and procedure - related complications ( 4.2 % versus 6.6 % ) compared to mechanical thrombectomy alone with a trend toward a higher revascularization rate ( 73 % versus 63 % ) and less mortality ( 27.7 % versus 40.1 % ) . although there was an association between time to reperfusion and clinical outcome after mechanical thrombectomy , its impact may not be as strong as iv thrombolysis , since a 40 % of late reperfused patients became independent . the second tested device for the recanalization of occluded intracranial arteries in acute ischemic stroke was the penumbra system . the initial safety trial was published in 2008 and , subsequently , was developed the penumbra pivotal stroke trial in 125 patients with cerebral ischemia up to 8-hours after symptoms onset , with neurological deficits defined by an nihss score 8 and an angiografically verified occlusion of a large intracranial vessel . . the recanalization rate was 81.6 % , sich occurred in 11.2 % and procedural events in 12.8 % of patients . all cause mortality was 32.8 % at 90 days with 25 % of patients achieving a modified rankin score 2 . similar to the merci and multimerci trials , good outcome were more frequent ( 29 % versus 9 % ) and mortality rate was lower ( 29 % versus 48 % ) with successful compared with unsuccessful recanalization . post - marketing experience of the penumbra system has shown a safety profile of the device comparable to the one reported in the pivotal trial with a trend to a better outcome ( 41 % versus 25 % ) . a systematic review and meta - analysis of 114 publications with 298 included patients treated with mechanical thrombectomy by diverse devices revealed a recanalization rate of 85 % and , in patients with accessible clots , 36 % of good outcome and 29 % of mortality . compared with a historical matched cohort for sex , age and nihss , patients who received mechanical intervention were 14.8 times more likely to have a long - term good outcome . a retrospective review of a prospective stroke database of endovascular treatment has shown that manual aspiration throughout large catheters added to other thrombolytic modalities increases recanalization rates with equivalent safety profile compared with other mechanical revascularization methods . removable cerebral stents and clot retriever devices referred to as stentrievers give a promising mechanical thrombectomy strategy . these devices avoid the need for strong antithrombotic medications that are used when an angioplasty with stenting is permanently deployed to achieve arterial revascularization , strategy that has been associated with an unacceptable risk of hemorrhagic complications . small case series and non controlled studies with different stent retriever devices ( solitaire ab / fr , trevo , revive ) have shown higher recanalization rates ( around in 90% of patients ) , shorter time to recanalization and a trend to improved outcomes and safety profile compared to other endovascular approaches [ 59 - 67 ] . moreover , swift and trevo2 trials showed the superiority of these new thrombectomy devices ( stentrievers ) compared with the first thrombectomy device used in stroke ( merci retriever ) in terms of recanalization rate and good neurologic outcome at 90 days [ 68 , 69 ] . consequently , retrievable stent - based devices are a newer generation of mechanical thrombectomy devices that represent a valuable tool for endovascular treatment of acute stroke since complete thrombus removal is safely achieved in many patients , within short time , increasing the potential for improved outcomes compared to other reperfusion treatments ( fig . have been investigated in the last years , only three controlled clinical trials of endovascular treatment in acute ischemic stroke have been conducted . the recent results of the interventional management of stroke iii ( ims - iii ) , synthesis expansion trial and the magnetic resonance and recanalization of stroke clots using embolectomy ( mr rescue ) have shown a lack of clinical benefit of endovascular therapy compared with standard medical therapy in patients with acute ischemic stroke . the ims - iii randomized patients treated with intravenous tpa within 3 hours after symptom onset to receive additional endovascular therapy or intravenous tpa alone . after 656 patients randomized , the study was halted due to futility according to the results of a pre - specified interim analysis . the proportion of participants with a modified rankin score of 2 or less at 90 days did not differ significantly according to treatement ( 40.8% with endovascular therapy and 38.7% with intravenous tpa ) . the synthesis randomized patients within 4.5 hours after symptoms onset to endovascular therapy or intravenous tpa . primary outcome was survival free of disability at 3 months ( defined as a modificed rankin score 0 or 1 ) . the results of this trial indicate that endovascular therapy is not superior to standard treatment with tpa showing that 30.4% patients in the endovascular - therapy group and 34.8% in the intravenous tpa group were alive without disability three months after stroke . in the mr rescue trial , patients within 8 hours after the onset of large - vessel , anterior - circulation strokes were randomly assigned to undergo mechanical embolectomy or receive standard care . despite the trial was designed to study whether brain imaging can identify patients who are most likely to benefit from therapies for acute ischemic stroke , among all patients mean scores on the modified rankin scale did not differ between embolectomy and standard care ( 3.9 vs 3.9 , p=0.99 ) groups . however , several limitations in the design and development of these previous trials that limit its generalizability have been reported [ 73 , 74 ] . ims - iii and synthesis trials have only focused on intravenous tpa eligible patients , and therefore their results can not be applied to intravenous tpa ineligible patients . furthermore , some of the patients randomized did not have arterial occlusion or have lesions with no or low chances of benefiting from endovascular therapy . mr rescue trial included patients with large infarct cores at baseline and with very low rates of adequate early reperfusion . moreover , the technology employed in the three trials is now obsolete . only a minimal fraction of the patients in ims - iii trial were treated with stentrivers and almost half were treated only with intra - arterial tpa . first - generation embolectomy devices were the second most commonly used modality . in synthesis trial , about two thirds of the patients were treated with intra - arterial tpa and fragmentation of the thrombus with a micro guide wire and in mr rescue trial embolectomy was performed with the first - generation merci or penumbra devices . overall , those reperfusion strategies do not reflect the current situation in which stent - retriever technology has demonstrated to achieve faster and more effective reperfusion compared to other strategies . despite endovascular treatment has not demonstrated a benefit in acute ischemic stroke in the three published trials , the safety of the treatment has not been questioned . given the limitations displayed , endovascular revascularization remains justified in selected patients with a large vascular occlusion , salvageable brain and employing stent - retriever devices to achieve a fast and effective reperfusion . thus aha / asa 2013 guidelines recommend intra - arterial treatment of acute mca occlusion within a 6-hour time window as an option ( class ii - i , level b ) and in patients with contraindications to the use of iv thrombolysis , such as recent surgery ( class ii , level c ) . another recommendation is that rescue intra - arterial fibrinolysis or mechanical thrombectomy approaches to recanalization in patients with large - artery occlusion who have not responded to intravenous fibrinolysis may be reasonable despite additional randomized trial data are needed ( class iib , level of evidence b ) . the use of stent retrievers such as solitaire fr and trevo are preferred to coil retrievers such as merci when mechanical thrombectomy is pursued ( class i ; level of evidence a ) . the results from the ongoing randomized clinical trials such as revascat ( clinicaltrials.gov identifier : nct01692379 ) , escape ( clinicaltrials.gov identifier : nct01778335 ) and swift prime ( clinicaltrials.gov identifier : nct01657461 ) comparing best medical therapy and endovascular treatment with novel designs according to current technologies will be crucial to elucidate the true effect of endovascular treatment on clinical outcomes in patients with acute large intracranial artery occlusions . intravenous thrombolysis has demonstrated to be safe and effective up to 4.5 hours after symptoms onset , however the frequency of treated patients is still quite low as it is recanalization rate obtained . endovascular approaches in the treatment of acute ischemic stroke offer higher recanalization rates compared with intravenous approach and has become a promising alternative for patients who are ineligible for intravenous thrombolysis or have failed in recanalyzing the occluded artery . nevertheless , its effectiveness in improving outcome has not been yet demonstrated in a randomized clinical trial . new randomized clinical trials with novel designs according to current technologies are necessary to elucidate the true effect of endovascular therapy for acute ischemic stroke on clinical outcomes . we disclose that this manuscript is an extended and updated version of our previously published manuscript ( curr cardiol rev 2010 ; 6 : 218 - 26 ) .

acute ischemic stroke is a major cause of morbidity and mortality in developed countries . intravenous thrombolysis with tissue plasminogen activator ( tpa ) within 4.5 hours of symptoms onset significantly improves clinical outcomes in patients with acute ischemic stroke . this narrow window for treatment leads to a small proportion of eligible patients to be treated . intravenous or intra - arterial trials , combined intravenous / intra - arterial trials , and newer devices to mechanically remove the clot from intracranial arteries have been investigated or are currently being explored to increase patient eligibility and to improve arterial recanalization and clinical outcome . new retrievable stent - based devices offer higher revascularization rates with shorter time to recanalization and are now generally preferred to first generation thrombectomy devices such as merci retriever or penumbra system . these devices have been shown to be effective for opening up occluded vessels in the brain but its efficacy for improving outcomes in patients with acute stroke has not yet been demonstrated in a randomized clinical trial . we summarize the results of the major systemic thrombolytic trials and the latest trials employing different endovascular approaches to ischemic stroke .

INTRODUTION INTRAVENOUS THROMBOLYSIS IN PATIENTS WITHIN 4.5 HOURS OF SYMPTOMS ONSET INTRAVENOUS THROMBOLYSIS IN PATIENTS WITH SALVAGEABLE TISSUE SELECTED BY MULTIMODAL NEUROIMAGING OTHER INTRAVENOUS THROMBOLYTIC AGENTS FOR ACUTE STROKE. ENDOVASCULAR TREATMENT IN ACUTE ISCHEMIC STROKE CONCLUSIONS CONFLICT OF INTEREST DISCLOSURES

PMC4237513

dengue is the most common arthropod - borne viral infection in the world and is estimated to transmit 390 million new infections every year . the genome of denv is comprised of a 10.7 kb , single , positive - stranded rna with at least four different circulating serotypes ( denv-1 to denv-4 ) . reportedly , a fifth serotype now complicates vaccine development . with increased levels of population growth , urbanization , and international travel , most of the current research on dengue infections is focused on the treatment of symptoms , which often can be a tedious and intensive process . as there is neither a drug nor any vaccine available to combat denv infections till date , it is highly important to explore and uncover small molecules that have potent anti - dengue activity . in the past half - decade these include an adenosine analog 1 , a n - sulfonylanthranilic acid derivate 2 , the chlorophenyl - thiophene derivate 3 , and most recently some 2,4-diaminoquinazoline derivatives 4 ( see fig . 1 ) . hence , further development of new chemical entities endowed with dengue inhibitory properties is warranted . our previous efforts herein , focused on tritylated and alkylated nucleoside analogs , resulting in compounds endowed with strong anti - flavivirus inhibitory properties . the five - membered imidazole ring is a structural unit found in many biologically active compounds . the strong therapeutic properties of imidazole containing drugs have encouraged medicinal chemists to synthesize a large number of novel chemotherapeutic agents comprising this entity . amongst others , imidazole core structures are found in different carboxypeptidase , hemeoxygenase and lactamase inhibitors , as well as among anti - inflammatory , anticancer , antibacterial , antifungal , antitubercular , antidiabetic and antiviral products , further highlighted with some examples . synthesized a series of novel 5-(nitro / bromo)-styryl-2-benzimidazole derivatives ( 5 ) and tested them for their antibacterial activity against staphylococcus aureus , escherichia coli and likewise evaluated the antifungal activity against candida albicans and aspergillus fumigates . et al . synthesized a series of 2-methylaminobenzimidazole derivatives in which compound 6 showed analgesic and anti - inflammatory activity comparable with the standard drug nimesulide . synthesized a series of 1 , 4-diarylimidazole-2(3h)-one derivatives 7 and their 2-thione analogs and found antitumor activity . synthesized imidazole derivatives for antiviral screening and different [ 2-(substituted phenyl)-imidazol-1-yl]-benzamides like 8 and 9 were selected as the most promising antiviral agents ( see fig . 2 ) . imidazole-4,5-dicarboxylic acid ( i45dc ) and its derivatives such as those bearing primary or secondary amides have previously been reported to be active against hiv-1 protease . likewise , some derivatives of this scaffold were uncovered as potential kinase inhibitors with antiproliferative activity against hl-60 cells . in addition , inhibition of protein protein interaction between hepatitis c glycoprotein e2 and cd81 has been reported previously . the i45dcs are known as well to be a structural component of some antibiotics and also to affect memory . substitution on the imidazole moiety herein has been reported , both in solution phase as well as in a combinatorial fashion on solid support . high throughput screening of a compound library led to the identification of n-(4-fluorophenyl)-n-(4-methyl-2-(3-methyl-1h - pyrazol-5-yl)phenyl)-1h - imidazole-4,5-dicarboxamide 15a ( see fig . 3 ) with an ec50 of 2.50 m and 3.47 m against denv and yfv , respectively . in search for new compounds with potential for clinical use as antiviral agents , a series of compounds based on this i45dc scaffold were synthesized with regiospecific attachment of the substituents and these analogs were investigated for their inhibitory properties against denv and yfv . in addition , the imidazole core was substituted for a pyrazine ring leading to a series of pyrazine-2,3-dicarboxylic acids ( p23dc ) . the intended substitution of the central imidazole ring would result in slightly different orientation of the attached amide groups , and in a change of the hydrogen bonding pattern exchanging the combination of a hydrogen donating and a hydrogen accepting nitrogen into two hydrogen accepting nitrogen atoms . in this communication , we report on the synthesis and biological activity of both new series of dissymmetric i45dcs and p23dcs , which we studied for their inhibitory properties against yfv and denv . the general procedure employed for the synthesis of dissymmetrically disubstituted i45dcs is shown in scheme 1 . imidazole-4,5-dicarboxylic acid 10 was allowed to reflux with socl2 in toluene with catalytic dmf to afford pyrazinedione diacid dichloride 11 in 92% yield . the literature route for amide formation suggests hydrolysis of the acid chlorides to carboxylic acids by the addition of water . amines are then added to open both acyl imidazole bonds affording two identical imidazole analogs . baures et al . , replaced the water with phenol , thereby modulating the reactivity of the acid chloride in comparison with an acyl imidazole bond for the subsequent addition of two different amines . the difficulty in preparing such analogs is that their synthesis is highly dependent upon the substituents to be introduced . primary benzyl amines , for example , are too reactive to provide selectivity in the reaction , whereas anilines can be added in a proper stoichiometric ratio in order to provide a pyrazine dione intermediate 12 which can often be purified by crystallization . in the case of aniline derivatives , the resulting pyrazine intermediates are often insoluble in the reaction solvent and can be isolated simply by vacuum filtration . in parallel , different bromoanilines or benzylamines were subsequently coupled with commercially available heterocyclic boronic acids through palladium catalyzed suzuki reaction to give the desired second aniline in 6075% yield . addition of this second aniline to the pyrazine intermediates 12a c resulted in opening of the acyl imidazole bond to obtain the expected product . based on this approach , a series of novel molecules was prepared within this i45dc family containing various substituted phenyl rings . for some of the final products 15 , the reaction sequence was changed with prior attachment of the aniline carrying a heterocycle . pyrazines are important pharmacophores present in a number of biologically active compounds such as antimycobacterial , antibacterial , antidiabetic , and hypnotic agents . to further explore previous structure activity relationship , we planned to substitute the five - membered imidazole ring with a pyrazine ring . hereto , regioselective functionalization of the pyrazine started from commercial pyrazine-2,3-dicarboxylic acid ( p23da ) and a four - step synthesis of the target compounds has been described in scheme 2 . p23da was converted to its corresponding anhydride 17 by treatment with acetic anhydride at room temperature . reaction with p - fluoroaniline or p - toluidine resulted in opening of the five - membered ring with concomitant formation of the first amide bond ( 18a , b ) . reaction with trifluoroacetic anhydride at 0 c afforded the anhydric activated product 19a or 19b allowing introduction of the second amide . hereto , the intermediate was reacted with preformed anilines or benzyl amines 14a m to give the target compounds 20a n in 6080% overall yields . all final products were purified by silica gel chromatography and characterized by h nmr , c nmr and hrms before evaluation of their inhibitory properties against denv and yfv . as shown in fig . 3 and schemes 1 and 2 , four main structural domains were chosen for optimization of hit 15a : the aromatic moiety ( ring a ) , the central pyrazine / imidazole core ( ring b ) , the substituted aromatic moiety ( ring c ) and the heterocyclic group ( ring d ) . these efforts have provided a few compounds that exhibited an improved biological profile with respect to the initial lead compound 15a . overall , improvement or reduction of the inhibitory activities for either denv or yfv is mostly running in parallel . the overall structure of 15a comprises an imidazole core carrying two aniline moieties coupled via an amide bond , with one of the aniline rings substituted with an additional heterocyclic ring ( ring d ) . therefore , our efforts to understand the structure activity relationship of the initial lead started with modifying the heterocyclic moiety as shown in table 1 . using p - fluoroaniline ( ring a ) and the i45dc core as a fixed fragment , substitution of the five - membered pyrazole moiety ( ring d ) with a thienyl or furanyl ring mostly led to considerable loss of activity ( i.e. , compare 15h , 15i , 15j versus 15a ) . however , activity remarkably was restored upon substitution of a methyl group for the para - fluorine in ring a ( 15k , 15l ) . the benzylamine substitution for ring a likewise annihilated the inhibitory properties ( 15 m ) , while in contrast a p - fluoro - phenylethylamine again displayed nice activity ( 15n ) . exchanging aniline ring c for a benzylamine preserved the activity when the supplementary heterocycle d was attached at the ortho position ( 15c , 15d ) , while para substitution led to considerable loss of activity ( 15e g ) within this context . a single attempt with an aminopyridine ring at site c proved inactive ( 15o ) . overall , the compounds 15b ( ec50 = 1.85 m , yfv ) , 15c ( ec50 = 1.93 m , denv ) , 15d ( ec50 = 2.61 m , yfv ) and 15k ( ec50 = 2.02 m , denv ) are endowed with the best inhibitory properties within this imidazole series . with these results in mind , we hoped to further improve the potency via replacement of the central five - membered imidazole ( ring b ) with a pyrazine scaffold , both rings containing two nitrogen atoms but providing a slightly different orientation for the aniline substituents . focusing on compound 15c as most potent representative for inhibition of denv ( ec50 = 1.92 m ) , we synthesized a series of pyrazine analogs with a pyridine substituent as the heterocyclic moiety d , attached at different positions of ring c. the inhibitory potency of these compounds was likewise examined against denv and yfv and is summarized in table 2 ( compounds 20a20n ) . unfortunately , the compound 20f corresponding to 15c , exhibited 20-fold decrease in activity against denv ( ec50 = 40.70 m ) and 6-fold against yfv ( ec50 = 21.29 m ) . concentrating on anti - denv activity , we noticed within the imidazole series that the para attachment of the heterocycle to ring c proved deleterious for the biological activity ( see 15e g ) . in contrast with these results , the para - pyrimidin-4-yl substituent on ring c proved advantageous within the pyrazine series and led to compound 20a displaying a 2-fold increase in anti - denv potency with ec50 of 0.93 m . a distinction however can be made having either a benzylamine ( n = 1 ) or an aniline ( n = 0 ) aromatic ring c. within the benzylamine series , the heterocycle is preferred at the para position with preference for concomitant presence of fluoroaniline at the a - site ( 20a , 20c ) over toluidine ( 20h , 20j ) . however , ortho attachment of the heterocycle on the benzylamine ring seriously reduced the activity ( 20f , 20 m ) . having an aniline ring at site c the results were less clear with mostly intermediate inhibitory properties ( 525 m ) , except for the strongly inhibiting compound 20b and the non - active analog 20e . within this pyrazine series ( 20a n ) the yfv inhibitory properties in general were slightly weaker compared to denv inhibition by these compounds . a series of tetracyclic inhibitors were prepared using i45cd and p23dc scaffolds and which further were part of a structureactivity investigation . the sar results of both series , i45dcs and p23dcs derivatives , as potent anti - dengue and anti - yellow fever virus agents are described above . our investigation led to the discovery of 15b , which is the most potent inhibitor of the series ( ec50 = 1.85 m ) against yfv , but followed closely in potency by the quite deviating structure 15d . the structural analog 15c on the other hand proved most inhibitory for dengue virus ( see fig . in addition , within the pyrazine series both compounds 20a and 20b were found to have potent denv inhibitory properties ( ec50 = 0.93 m ) uncovered to date . key features of these inhibitors include the central bis - amide arrangement which is found to be a metal - chelating component in many drugs . in summary , determination of the antiviral activities shows that i45dc and p23dc are attractive candidate scaffolds for further studies and the interesting activities observed so far , warrant an in depth study of their mechanism of action . all other chemicals were provided by aldrich or acros and were of the highest quality . h and c nmr spectra were determined with a 300 mhz varian gemini apparatus with tetramethylsilane as internal standard for the h nmr spectra ( s = singlet , d = doublet , dd = double doublet , t = triplet , br . d = broad doublet , m = multiplet ) and the solvent signal dmso - d6 ( = 39.6 ppm ) or cdcl3 ( = 76.9 ppm ) for the c nmr spectra . exact mass measurements were performed with a quadrupole / orthogonal acceleration time - of - flight tandem mass spectrometer ( qtof2 , micromass , manchester , uk ) fitted with a standard electrospray ionization ( esi ) interface . all solvents were carefully dried or bought as such . to a dry round - bottom flask was added 1.0 g of imidazole-4,5-dicarboxylic acid ( 10 , 1 mmol ) and 10 ml of toluene . to this stirred suspension were added 3.0 ml of thionyl chloride ( 6.5 mmol ) and 0.250 ml of dmf ( catalytic ) . the resulting mixture was refluxed for 16 h. after the mixture was cooled in ice bath , the solid product was collected by vacuum filtration , washed with two 20 ml portions of toluene , and dried under vacuum to yield 0.95 g of crude 11 ( yield : 95% ) as a yellow solid . this product was used further without characterization . to a suspension of 11 ( 1.0 mmol ) in 10 ml of dichloromethane was added n , n - diethylaniline ( 2 mmol ) , and corresponding substituted aniline ( 1.7 mmol ) at 10 c . the reaction mixture was stirred at room temperature for 35 h before collecting highly insoluble colored solid 12a the product was washed with portions of 10 ml dcm , cold water , and acetone , respectively . note : addition of excess of aniline results in opening of acyl imidazole bonds affording unwanted products . yield : 85% ; orange solid ; h nmr ( 300 mhz , cdcl3 ) : 10.74 ( s , 2h ) , 9.09 ( s , 2h ) , 7.80 ( d , 4h , j = 7.8 hz ) , 7.41 ( t , 4h , j = 8.1 hz ) , 7.17 ( t , 2h , j = 7.5 hz ) . c nmr ( 75 mhz , cdcl3 ) : 162.3 , 158.1 , 149.2 , 145.2 , 138.7 , 138.2 , 129.0 , 124.5 , 120.1 . yield : 87% ; yellow solid ; h nmr ( 300 mhz , cdcl3 ) : 10.76 ( s , 2h ) , 9.09 ( s , 2h ) , 7.857.81 ( m , 4h ) , 7.25 ( m , 4h , j = 9.0 hz ) . c nmr ( 75 mhz , cdcl3 ) : 160.3 , 158.1 , 157.2 , 149.0 , 145.0 , 138.6 , 134.8 , 134.8 , 122.1 , 122.0 , 120.7 , 115.5 . hrms calcd for c22h15n6o4 [ m+h ] : 427.1149 ; found : 427.1151 . yield : 89% ; red solid ; h nmr ( 600 mhz , dmso ) : 10.62 ( s , 2h ) , 9.07 ( s , 2h ) , 7.67 ( d , 4h , j = 8.4 hz ) , 7.20 ( d , 4h , j = 7.8 hz ) , 2.30 ( s , 6h ) . c nmr ( 125 mhz , dmso ) : 157.8 , 149.2 , 145.2 , 138.6 , 135.9 , 133.5 , 129.4 , 120.4 , 120.0 , 20.6 . yield : 74% ; red solid ; h nmr ( 300 mhz , dmso ) : 10.01 ( s , 2h ) , 8.97 ( s , 2h ) , 7.797.63 ( m , 6h ) , 7.357.23 ( m , 6h ) , 2.37 ( s , 6h , 2ch3 ) . hrms calcd for c32h23n6o4s2 [ m+h ] : 619.1237 ; found : 619.1238 . in view of insufficient solubility c nmr yield : 72% ; yellow solid ; h nmr ( 300 mhz , dmso ) : 10.46 ( s , 2h ) , 9.07 ( s , 2h ) , 7.817.61 ( m , 6h ) , 7.256.63 ( m , 6h ) , 2.38 ( s , 6h , 2ch3 ) . hrms calcd for c32h23n6o6 [ m+h ] : 587.1673 ; found : 587.1672 . in view of insufficient solubility c nmr to a solution of corresponding aniline / benzyl amine ( 1.0 mmol ) in 10 ml dioxane : water ( 1:1 ) , was added anhydrous k2co3 ( 1.5 mmol ) , aryl boronic acids ( 1.2 mmol ) and pd(tpp)2cl2 ( 0.025 mmol ) in a seal tube . the mixture was purged with argon for 30 min at rt and heated at 100 c for 1 h in microwave . after completion , the solvent was evaporated under reduced pressure and the residue was purified by column chromatography on a silica gel to give the desired product . yield : 52% ; white solid ; h nmr ( 300 mhz , cdcl3 ) : 7.50 ( d , 1h , j = 7.2 hz ) , 7.13 ( t , 1h , j = 7.5 hz ) , 6.796.75 ( m , 2h ) , 6.39 ( s , 1h ) , 2.37 ( s , 3h , ch3 ) . yield : 62% ; yellow oil ; h nmr ( 300 mhz , cdcl3 ) : 8.70 ( s , 1h ) , 8.618.59 ( m , 1h ) , 7.837.80 ( m , 1h),7.407.35 ( m , 1h ) , 7.057.01 ( m , 1h ) , 6.94 ( m , 1h ) , 6.736.71 ( d , 1h , j = 8.1 hz ) , 3.60 ( bs , 2h , nh2 ) , 2.30 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 149.2 , 147.4 , 140.3 , 135.7 , 130.0 , 129.0 , 127.3 , 122.6 , 115.2 , 19.5 . yield : 59% ; white solid ; h nmr ( 300 mhz , cdcl3 ) : 8.698.67 ( m , 2h ) , 7.447.42 ( m , 2h ) , 7.066.96 ( m , 2h ) , 6.72 ( d , 1h , j = 8.1 hz ) , 3.69 ( bs , 2h , nh ) , 2.30 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 150.4 , 130.6 , 130.5 , 124.0 , 116.4 , 20.5 . yield : 72% ; yellow solid ; h nmr ( 300 mhz , cdcl3 ) : 8.73 ( s , 1h ) , 8.628.60 ( m , 1h ) , 7.847.80 ( m , 1h ) , 7.417.36 ( m , 1h ) , 7.247.11 ( m , 2h ) , 6.896.79 ( m , 2h ) , 3.75 ( bs , 2h , nh2 ) . c nmr ( 75 mhz , cdcl3 ) : 150.2 , 148.6 , 143.9 , 136.6 , 135.4 , 130.7 , 129.5 , 123.6 , 119.0 , 116.0 . yield : 69% ; white solid ; h nmr ( 300 mhz , cdcl3 ) : 8.698.67 ( m , 2h ) , 7.447.28 ( m , 2h ) , 7.257.13 ( m , 2h ) , 6.896.78 ( m , 2h ) , 3.82 ( bs , 2h , nh2 ) . c nmr ( 75 mhz , cdcl3 ) : 149.4 , 129.2 , 128.8 , 123.0 , 118.0 , 115.2 . yield : 55% ; white solid ; h nmr ( 300 mhz , cdcl3 ) : 8.86 ( m , 2h ) , 8.618.59 ( m , 1h ) , 7.917.87 ( m , 1h ) , 7.607.57 ( m , 2h ) , 7.477.40 ( m , 2h ) , 7.387.36 ( m , 1h ) , 3.96 ( s , 2h , ch2 ) , 1.62 ( bs , 2h , nh2 ) . c nmr ( 75 mhz , cdcl3 ) : 149.9 , 127.5 , 126.8 , 121.1 , 45.7 . yield : 59% ; white solid ; h nmr ( 300 mhz , cdcl3 ) : 8.648.62 ( m , 2h ) , 7.617.41 ( m , 6h ) , 3.92 ( s , 2h , ch2 ) , 1.77 ( bs , 2h , nh2 ) . c nmr ( 75 mhz , cdcl3 ) : 149.2 , 126.9 , 126.2 , 120.5 , 45.1 . yield : 65% ; white solid ; h nmr ( 300 mhz , cdcl3 ) : 8.768.74 ( m , 1h ) , 7.567.10 ( m , 8h ) , 3.92 ( s , 2h , ch2 ) . c nmr ( 75 mhz , cdcl3 ) : 149.1 , 132.0 , 131.3 , 131.1 , 131.1 , 128.2 , 127.7 , 127.6 , 127.5 , 126.9 , 122.7 , 120.5 , 46.0 . yield : 62% ; off - white solid ; h nmr ( 300 mhz , cdcl3 ) : 7.477.40 ( m , 2h ) , 7.337.31 ( m , 1h ) , 7.09 ( s , 1h ) , 7.037.00 ( m , 1h ) , 6.756.72 ( d , 1h , j = 8.1 hz ) , 3.73 ( bs , 2h , nh2 ) , 2.33 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 141.5 , 140.1 , 130.7 , 129.1 , 128.5 , 127.8 , 126.0 , 122.6 , 122.5 , 116.0 , 20.5 . hrms calcd for c11h12n1s1 [ m+h ] : 190.0685 ; found : 190.0686 . yield : 58% ; off - white solid ; h nmr ( 300 mhz , cdcl3 ) : 7.617.58 ( m , 2h ) , 7.477.28 ( m , 6h ) , 3.92 ( s , 2h , ch2 ) . c nmr ( 75 mhz , cdcl3 ) : 127.7 , 126.8 , 126.5 , 126.3 , 120.2 , 46.3 . yield : 52% ; white solid ; h nmr ( 300 mhz , cdcl3 ) : 7.587.12 ( m , 6h ) , 3.91 ( s , 2h , ch2 ) . c nmr ( 150 mhz , cdcl3 ) : 162.1 , 160.8 , 160.5 , 142.0 , 141.1 , 135.8 , 132.7 , 130.1 , 129.8 , 129.3 , 129.0 , 128.8 , 128.6 , 128.4 , 128.2 , 127.7 , 127.5 , 127.4 , 127.1 , 127.0 , 126.8 , 125.5 , 125.0 , 123.4 , 122.7 , 46.8 , 44.2 . yield : 63% ; white solid ; h nmr ( 300 mhz , cdcl3 ) : 7.52 ( s , 1h ) , 7.32 ( s , 1h ) , 6.976.94 ( m , 1h ) , 6.716.52 ( m , 3h ) , 2.30 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 152.5 , 141.2 , 140.8 , 129.5 , 127.8 , 127.6 , 116.9 , 116.2 , 111.3 , 106.3 , 20.4 . hrms calcd for c11h12n1o1 [ m+h ] : 174.0913 ; found : 174.0910 . yield : 57% ; white solid ; h nmr ( 300 mhz , cdcl3 ) : 7.84 ( s , 1h ) , 7.28 ( s , 1h ) , 6.99 ( d , j = 3.3 hz , 1h ) , 6.736.72 ( m , 1h ) , 4.96 ( bs , 2h , nh2 ) , 2.49 ( s , 3h , ch3 ) , 2.18 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 153.6 , 146.5 , 139.8 , 138.5 , 137.1 , 125.6 , 125.5 , 122.7 , 114.5 , 17.0 , 14.9 . to a stirred solution of 12a e ( 1.0 mmol ) in 10 ml dcm , was added dipea ( 3.0 mmol ) , and 14a m ( 3.0 mmol ) under argon atmosphere at room temperature . the mixture was allowed to reflux for 1648 h. two alternative workup procedures were followed depending on whether the product precipitated out of solution or not . workup 1 ( for soluble products ) : the dcm was evaporated using a rotavapor . the product was purified by flash chromatography on silica gel . workup 2 ( for insoluble products ) : the precipitate was collected by filtration using a buchner funnel and washed on the frit with dcm ( 2025 ml ) until the yellow impurities had disappeared . yield : 86% ; h nmr ( 500 mhz , dmso ) : 13.69 ( bs , 1h , nh ) , 13.30 ( bs , 1h , nh ) , 13.05 ( bs , 1h , nh ) , 12.78 ( bs , 1h , nh ) , 8.65 ( d , 1h , j = 8.0 hz ) , 7.96 ( s , 1h ) , 7.787.23 ( m , 9h ) , 6.54 ( s , 1h ) , 2.31 ( s , 3h , ch3 ) . c nmr ( 125 mhz , dmso ) : 163.3 , 159.6 , 158.0 , 156.4 , 150.3 , 140.2 , 137.3 , 135.2 , 135.0 , 133.9 , 130.0 , 128.7 , 124.8 , 123.2 , 121.9 , 121.6 , 116.4 , 116.2 , 103.1 , 10.8 . yield : 83% ; h nmr ( 600 mhz , dmso ) : 8.76 ( d , 0.5h , j = 6.6 hz ) , 7.967.17 ( m , 10h ) , 4.57 ( d , 2h , j = 6.0 hz , ch2 ) . c nmr ( 150 mhz , dmso ) : 159.5 , 157.9 , 150.0 , 137.4 , 127.1 , 134.8 , 132.7 , 132.4 , 131.8 , 131.7 , 129.3 , 129.1 , 129.0 , 128.7 , 128.1 , 122.4 , 122.0 , 116.0 , 115.8 , 43.1 . yield : 86% ; h nmr ( 600 mhz , dmso ) : 13.53 ( bs , 0.5h , nh ) , 13.45 ( bs , 0.5h , nh ) , 9.40 ( bs , 0.5h , nh ) , 9.23 ( bs , 0.5h , nh ) , 7.997.18 ( m , 12h ) , 4.58 ( d , 2h , j = 6.6 hz , ch2 ) . c nmr ( 150 mhz , dmso ) : 164.3 , 156.1 , 140.5 , 137.3 , 137.0 , 132.7 , 132.5 , 129.9 , 129.0 , 128.0 , 127.6 , 127.1 , 126.3 , 123.7 , 121.1 , 115.9 , 115.7 , 42.9 . hrms calcd for c22h16f1n4o2s1 [ mh ] : 419.0973 ; found : 419.0967 . yield : 76% ; h nmr ( 600 mhz , dmso ) : 13.55 ( s , 1h , nh ) , 13.49 ( bs , 1h , nh ) , 10.49 ( s , 1h ) , 10.06 ( s , 1h ) , 9.47 ( s , 1h ) , 8.857.20 ( m , 9h ) , 4.59 ( d , 2h , j = 6.6 hz , ch2 ) . c nmr ( 150 mhz , dmso ) : 164.2 , 156.4 , 148.6 , 147.7 , 139.3 , 137.2 , 136.0 , 135.6 , 135.1 , 134.4 , 133.0 , 128.9 , 128.4 , 128.3 , 127.3 , 127.2 , 124.2 , 121.4 , 121.4 , 116.1 , 116.9 , 42.3 . hrms calcd for c23h17f1n5o2 [ mh ] : 414.1372 ; found : 414.1372 . yield : 78% ; h nmr ( 300 mhz , dmso ) : 9.84 ( bs , 1h , nh ) , 7.97 ( s , 1h ) , 7.877.19 ( m , 15h ) , 4.57 ( d , 2h , j = 6.3 hz , ch2 ) . c nmr ( 75 mhz , dmso ) : 160.1 , 157.0 , 141.4 , 141.4 , 138.0 , 137.0 , 134.9 , 134.0 , 128.1 , 128.0 , 127.6 , 127.1 , 126.3 , 126.0 , 121.7 , 121.6 , 120.9 , 120.7 , 115.9 , 115.6 , 42.2 . yield : 81% ; h nmr ( 600 mhz , dmso ) 13.56 ( s , 1h , nh ) , 13.47 ( bs , 1h , nh ) , 9.50 ( s , 1h , nh ) , 8.617.20 ( m , 13h ) , 4.59 ( d , 2h , j = 6.0 hz , ch2 ) . c nmr ( 150 mhz , dmso ) : 164.2 , 156.2 , 150.5 , 150.3 , 146.9 , 140.4 , 137.1 , 135.9 , 135.0 , 133.0 , 128.8 , 128.3 , 128.2 , 127.1 , 127.0 , 121.3 , 121.2 , 116.0 , 115.9 , 42.2 . hrms calcd for c23h17f1n5o2 [ mh ] : 414.1372 ; found : 414.1369 . yield : 89% ; h nmr ( 500 mhz , dmso ) : 13.64 ( s , 0.7h , nh ) , 13.56 ( s , 0.3h , nh ) , 13.22 ( s , 0.7h , nh ) , 12.44 ( s , 0.3h , nh ) , 10.46 ( s , 0.3h , nh ) , 10.12 ( s , 0.7h , nh ) , 7.997.15 ( m , 13h ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 125 mhz , dmso ) : 162.5 , 156.0 , 138.3 , 137.1 , 136.7 , 135.2 , 134.9 , 132.9 , 131.5 , 130.7 , 130.6 , 130.0 , 129.2 , 128.6 , 128.6 , 128.4 , 128.4 , 127.0 , 126.2 , 124.9 , 124.2 , 124.0 , 122.9 , 199.9 , 121.2 , 121.2 , 116.0 , 115.8 , 115.2 , 115.1 , 20.6 . yield : 77% ; h nmr ( 300 mhz , dmso ) : 13.69 ( bs , 1h , nh ) , 13.20 ( bs , 1h , nh ) , 10.60 ( s , 1h , nh ) , 8.057.55 ( m , 6h ) , 7.276.56 ( m , 5h ) , 2.38 ( s , 3h , ch3 ) . yield : 81% ; h nmr ( 600 mhz , dmso ) : 13.63 ( s , 0.7h , nh ) , 13.56 ( s , 0.3h , nh ) , 13.18 ( s , 0.7h , nh ) , 12.47 ( s , 0.3h , nh ) , 10.34 ( s , 0.3h , nh ) , 10.12 ( s , 0.7h , nh ) , 7.967.12 ( m , 12h ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 125 mhz , dmso ) : 162.4 , 156.0 , 138.5 , 138.3 , 137.1 , 135.2 , 132.8 , 131.5 , 130.6 , 129.9 , 129.3 , 128.6 , 128.4 , 127.0 , 124.2 , 124.0 , 120.9 , 119.4 , 20.6 . yield : 79% ; h nmr ( 600 mhz , dmso ) : 13.59 ( s , 0.7h , nh ) , 13.54 ( s , 0.3h , nh ) , 13.09 ( s , 0.3h , nh ) , 12.52 ( s , 0.7h , nh ) , 10.26 ( s , 0.3h , nh ) , 10.10 ( s , 0.7h , nh ) , 7.987.18 ( m , 10h ) , 2.36 ( s , 3h , ch3 ) , 2.28 ( s , 3h , ch3 ) . c nmr ( 125 mhz , dmso ) : 162.4 , 155.8 , 138.3 , 137.0 , 136.0 , 135.1 , 133.1 , 132.7 , 131.5 , 130.6 , 129.9 , 129.6 , 129.4 , 129.0 , 128.6 , 128.5 , 128.4 , 128.4 , 127.0 , 124.2 , 124.0 , 120.9 , 119.4 , 20.6 . hrms calcd for c23h21n4o2s1 [ m+h ] : 417.1380 ; found : 417.1377 . yield : 76% ; 13.58 ( s , 0.7h , nh ) , 13.52 ( s , 0.3h , nh ) , 13.07 ( s , 0.3h , nh ) , 12.50 ( s , 0.7h , nh ) , 10.23 ( s , 0.3h , nh ) , 10.08 ( s , 0.7h , nh ) , 7.977.93 ( m , 2h ) , 7.717.56 ( m , 4h ) , 7.337.11 ( m , 5h ) , 2.35 ( s , 3h , ch3 ) , 2.28 ( s , 3h , ch3 ) . c nmr ( 125 mhz , dmso ) : 162.4 , 155.8 , 138.3 , 136.9 , 136.0 , 135.1 , 133.1 , 132.7 , 131.5 , 130.6 , 129.8 , 129.6 , 129.4 , 129.0 , 128.6 , 128.5 , 128.4 , 128.4 , 127.0 , 124.2 , 124.0 , 120.9 , 119.4 , 20.6 , 20.6 . hrms calcd for c23h21n4o3 [ m+h ] : 401.1608 ; found : 401.1602 . yield : 83% ; h nmr ( 300 mhz , cdcl3 ) : 11.84 ( bs , 1h , nh ) , 11.57 ( bs , 1h , nh ) , 10.32 ( bs , 1h , nh ) , 8.16 ( d , 1h , j = 8.4 hz ) , 7.617.18 ( m , 10h ) , 6.74 ( d , 1h , j = 3.3 hz ) , 6.576.55 ( m , 1h ) , 4.70 ( d , 2h , j = 6.0 hz , ch2 ) , 2.40 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 161.7 , 159.0 , 142.4 , 134.9 , 134.8 , 130.5 , 129.1 , 128.6 , 127.9 , 127.4 , 127.2 , 123.0 , 111.7 , 108.5 , 43.5 , 21.0 . yield : 75% ; h nmr ( 300 mhz , cdcl3 ) : 11.13 ( bs , 1h , nh ) , 10.32 ( bs , 1h , nh ) , 8.18 ( d , 1h , j = 8.7 hz ) , 7.676.55 ( m , 10h ) , 3.73 ( m , 2h , ch2 ) , 3.00 ( t , 2h , j = 7.5 hz ch2 ) , 2.42 ( s , 3h , ch3 ) . c nmr ( 150 mhz , dmso ) : 161.5 , 157.9 , 157.1 , 150.6 , 150.3 , 143.1 , 142.8 , 142.3 , 142.2 , 138.4 , 137.4 , 136.8 , 136.5 , 135.7 , 135.0 , 133.3 , 132.6 , 130.4 , 130.2 , 129.0 , 128.7 , 128.3 , 127.6 , 127.3 , 127.1 , 125.5 , 125.0 , 124.5 , 123.8 , 118.0 , 115.7 , 115.4 , 113.1 , 112.8 , 112.1 , 109.1 , 108.9 , 77.1 , 60.9 , 34.81 . hrms calcd for c24h22fn4o3 [ m+h ] : 433.1670 ; found : 433.1668 . yield : 76% ; h nmr ( 600 mhz , cdcl3 ) : 13.63 ( bs , 1h , nh ) , 13.08 ( bs , 0.7h , nh ) , 12.92 ( bs , 0.3h , nh ) , 10.70 ( bs , 0.7h , nh ) , 10.54 ( bs , 0.3h , nh ) , 8.317.08 ( m , 8h ) , 6.796.76 ( m , 1h ) , 2.39 ( s , 3h , ch3 ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 150 mhz , cdcl3 ) : 184.6 , 149.5 , 147.4 , 144.6 , 143.7 , 141.2 , 138.3 , 138.2 , 137.4 , 137.3 , 135.6 , 132.9 , 129.3 , 129.2 , 129.1 , 128.5 , 127.1 , 126.8 , 126.3 , 128.5 , 17.5 , 15.1 . hrms calcd for c22h19n5o2s1 [ mh ] : 416.1187 ; found : 416.1187 . sodium dodecyl sulfate ( sds , 6 mmol ) was added to a stirred heterogeneous suspension of amine ( 5 mmol ) in water ( 20 ml ) until a homogeneous solution was formed , ( in case of turbidity , the mixture was warmed to obtain a clear solution ) . anhydride 17 ( 5 mmol ) dissolved in acetonitrile ( 5 ml ) was added to this solution in one lot . after stirring for 1 h at room temperature , the acetonitrile was evaporated and the product precipitated from the aqueous layer . to the aqueous solution containing precipitate , solid sodium hydrogen carbonate was added pinch - wise until the effervescence ceased and the ph was 6.0 . the remaining precipitated product was filtered , washed with water ( 20 ml ) , dried in a vacuum desiccator . in cases where the product did not precipitate , the reaction mixture was extracted with ethyl acetate ( 2 25 ml ) . the combined organic extracts were dried with anhydrous na2so4 and the solvent was removed in a rotary evaporator under reduced pressure to yield the pure product . yield : 72% ; white solid ; h nmr ( 300 mhz , dmso ) : 10.86 ( bs , 1h ) , 8.90 ( s , 2h ) , 7.837.78 ( m , 2h ) , 7.22 ( t , 2h , j = 9.0 hz ) . yield : 65% ; white solid ; h nmr ( 300 mhz , dmso ) : 10.68 ( bs , 1h ) , 8.88 ( s , 2h ) , 7.67 ( d , j = 8.4 hz , 2h ) , 7.17 ( t , j = 8.4 hz , 2h ) , 2.29 ( s , 3h , ch3 ) . c nmr ( 75 mhz , dmso ) : 166.5 , 162.3 , 146.5 , 145.8 , 145.6 , 144.6 , 136.0 , 133.4 , 129.3 , 120.2 , 20.7 . trifluoroacetic anhydride ( 1.5 equiv ) was added dropwise to a stirring solution of acid 18a b ( 1 equiv ) and et3n ( tea , 3 equiv ) in 1,4-dioxane ( 20 ml ) that was kept at 0 c with an ice bath . after 15 min the yellow solution was allowed to warm to room temperature and was stirred for 30 min , then it was poured in cold h2o ( 100 ml ) . a precipitate formed , which was collected by filtration using a buchner funnel and washed with h2o . yield : 70% ; off - white solid ; h nmr ( 300 mhz , cdcl3 ) : 9.06 ( d , 2h , j = 5.1 hz ) , 7.737.69 ( m , 2h ) , 7.15 ( t , j = 8.4 hz , 2h ) . c nmr ( 75 mhz , cdcl3 ) : 150.9 , 149.5 , 128.7 , 128.6 , 116.2 , 116.0 . yield : 66% ; white solid ; h nmr ( 300 mhz , cdcl3 ) : 9.079.03 ( m , 2h ) , 7.697.28 ( m , 4h ) , 2.43 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 150.5 , 149.0 , 129.5 , 126.4 , 21.0 . phthalisoimide 19a b ( 1 equiv ) was added to stirring solution of the aniline / benzylamine 14a m ( 1.2 equiv ) in thf . the mixture was diluted with etoac ( triple the volume of thf ) and washed three times with 1 m hcl . yield : 76% ; h nmr ( 300 mhz , dmso ) : 10.65 ( bs , 1h , nh ) , 9.45 ( t , j = 6.6 hz , 1h , nh ) , 8.88 ( q , 2h , j = 2.4 hz ) , 8.648.61 ( m , 2h ) , 7.787.69 ( m , 6h ) , 7.50 ( d , j = 8.4 hz , 2h ) , 7.21 ( t , j = 9.0 hz , 2h ) , 2.39 ( d , j = 6.3 hz , 2h , ch2 ) . c nmr ( 75 mhz , dmso ) : 164.0 , 163.7 , 160.1 , 156.9 , 150.3 , 148.3 , 146.9 , 145.4 , 145.3 , 144.5 , 140.5 , 135.8 , 135.5 , 128.3 , 126.9 , 121.7 , 121.6 , 121.2 , 115.7 , 115.4 , 42.2 . yield : 74% ; h nmr ( 300 mhz , cdcl3 ) : 9.02 ( bs , 1h , nh ) , 8.698.54 ( m , 5h ) , 8.27 ( d , j = 8.4 hz , 1h ) , 7.84 ( d , j = 8.1 hz , 1h ) , 7.707.66 ( m , 2h ) , 7.367.26 ( m , 2h ) , 7.117.03 ( m , 3h ) , 2.39 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 161.8 , 161.5 , 150.1 , 149.1 , 144.3 , 137.3 , 135.7 , 134.2 , 131.8 , 131.0 , 130.1 , 123.7 , 123.2 , 122.2 , 122.1 , 116.1 , 115.8 , 21.1 . hrms calcd for c24h19f1n5o2 [ m+h ] : 428.1517 ; found : 428.1514 . yield : 72% ; h nmr ( 300 mhz , cdcl3 ) : 9.33 ( bs , 1h , nh ) , 8.76 ( bs , 1h , nh ) , 8.63 ( s , 2h ) , 8.548.52 ( d , j = 4.8 hz , 1h ) , 7.867.27 ( m , 9h ) , 7.05 ( m , j = 8.7 hz , 2h ) , 4.76 ( d , j = 6.0 hz , 2h , ch2 ) . c nmr ( 75 mhz , cdcl3 ) : 164.9 , 161.6 , 161.5 , 148.6 , 148.3 , 147.6 , 146.1 , 144.6 , 144.0 , 137.8 , 137.3 , 136.3 , 134.4 , 133.6 , 128.8 , 127.6 , 123.7 , 122.1 , 122.0 , 116.0 , 115.7 , 43.7 . hrms calcd for c24h19f1n5o2 [ m+h ] : 428.1517 ; found : 428.1517 . yield : 66% ; h nmr ( 300 mhz , cdcl3 ) : 10.72 ( bs , 1h , nh ) , 10.27 ( bs , 1h , nh ) , 8.908.86 ( m , 2h ) , 8.588.56 ( m , 2h ) , 7.857.76 ( m , 3h ) , 7.557.37 ( m , 5h ) , 7.22 ( t , j = 9.0 hz , 2h ) . c nmr ( 75 mhz , cdcl3 ) : 163.2 , 163.1 , 149.8 , 147.0 , 146.2 , 145.1 , 144.9 , 135.2 , 134.4 , 133.6 , 130.3 , 129.3 , 126.6 , 126.2 , 124.0 , 121.9 , 121.8 , 115.7 , 115.4 , 79.3 . hrms calcd for c23h17f1n5o2 [ m+h ] : 414.1361 ; found : 414.1363 . yield : 76% ; h nmr ( 300 mhz , cdcl3 ) : 9.04 ( bs , 1h , nh ) , 8.78 ( bs , 1h , nh ) , 8.708.56 ( m , 4h ) , 8.45 ( d , j = 8.1 hz , 1h ) , 7.87 ( d , j = 7.8 hz , 1h ) , 7.727.69 ( m , 2h ) , 7.527.7.28 ( m , 4h ) , 7.07 ( t , j = 8.7 hz , 2h ) . c nmr ( 75 mhz , cdcl3 ) : 161.7 , 161.5 , 150.1 , 149.2 , 144.3 , 137.3 , 134.4 , 134.0 , 133.5 , 130.5 , 129.9 , 129.5 , 125.8 , 123.7 , 123.1 , 122.2 , 122.1 , 116.1 , 115.8 . hrms calcd for c23h17f1n5o2 [ m+h ] : 414.1361 ; found : 414.1365 . yield : 68% ; h nmr ( 600 mhz , dmso ) : 10.67 ( bs , 1h , nh ) , 9.41 ( t , j = 6.0 hz , 1h ) , 8.918.89 ( m , 2h ) , 7.757.19 ( m , 8h ) , 4.51 ( d , j = 6.0 hz , 2h ) . c nmr ( 125 mhz , dmso ) : 164.3 , 163.6 , 159.2 , 157.6 , 150.6 , 149.0 , 148.1 , 145.3 , 144.5 , 137.4 , 135.3 , 132.4 , 129.0 , 128.5 , 127.8 , 122.1 , 121.5 , 115.5 , 115.4 , 42.9 . yield : 76% ; h nmr ( 300 mhz , cdcl3 ) : 8.95 ( bs , 1h , nh ) , 8.668.58 ( m , 5h ) , 8.32 ( d , j = 8.4 hz , 1h ) , 7.727.67 ( m , 2h ) , 7.43 ( d , j = 8.4 hz , 2h ) , 7.30 ( d , j = 8.4 hz , 2h ) , 7.137.7.04 ( m , 3h ) , 2.40 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 150.0 , 145.9 , 144.0 , 143.8 , 130.9 , 130.0 , 129.9 , 124.0 , 122.6 , 121.7 , 121.6 , 115.6 , 115.3 , 20.6 . yield : 78% ; h nmr ( 300 mhz , cdcl3 ) : 9.19 ( bs , 1h , nh ) , 8.668.62 ( m , 4h ) , 7.647.47 ( m , 8h ) , 7.17 ( d , j = 8.1 hz , 3h ) , 4.78 ( d , j = 6.0 hz , 2h , ch2 ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 165.1 , 161.2 , 150.4 , 148.1 , 148.0 , 145.8 , 144.8 , 143.8 , 139.0 , 137.6 , 134.9 , 134.8 , 129.7 , 128.8 , 127.5 , 121.7 , 120.3 , 43.7 , 21.1 . hrms calcd for c25h22n5o2 [ m+h ] : 424.1768 ; found : 424.1765 . yield : 76% ; h nmr ( 300 mhz , cdcl3 ) : 8.95 ( bs , 1h , nh ) , 8.728.56 ( m , 5h ) , 8.33 ( d , j = 8.1 hz , 1h ) , 7.89 ( d , j = 7.5 hz , 1h ) , 7.62 ( d , j = 8.1 hz , 2h ) , 7.387.14 ( m , 4h ) , 2.42 ( s , 3h , ch3 ) , 2.37 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 150.2 , 149.2 , 144.5 , 144.1 , 137.3 , 135.5 , 134.8 , 134.2 , 131.9 , 131.0 , 130.1 , 123.6 , 123.3 , 120.3 , 21.1 . hrms calcd for c25h22n5o2 [ m+h ] : 424.1768 ; found : 424.1767 . yield : 71% ; h nmr ( 300 mhz , cdcl3 ) : 9.24 ( bs , 1h , nh ) , 8.788.54 ( m , 4h ) , 7.887.84 ( m , 1h ) , 7.63 ( d , j = 8.4 hz , 2h ) , 7.567.17 ( m , 8h ) , 4.79 ( d , j = 6.0 hz , 2h , ch2 ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 165.2 , 161.2 , 148.6 , 148.3 , 148.1 , 145.7 , 144.6 , 143.8 , 137.9 , 137.2 , 136.4 , 134.9 , 134.7 , 134.4 , 129.7 , 128.9 , 127.6 , 123.7 , 120.3 , 43.7 , 21.1 . yield : 72% ; h nmr ( 300 mhz , cdcl3 ) : 8.95 ( bs , 1h , nh ) , 8.688.51 ( m , 6h ) , 7.63 ( d , j = 8.4 hz , 2h ) , 7.507.19 ( m , 7h ) , 2.37 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 150.5 , 146.2 , 144.6 , 144.1 , 134.9 , 134.1 , 129.9 , 129.8 , 125.7 , 124.5 , 123.0 , 120.3 , 21.1 . hrms calcd for c24h20n5o2 [ m+h ] : 410.1611 ; found : 410.1608 . yield : 68% ; h nmr ( 300 mhz , cdcl3 ) : 8.70 ( bs , 1h , nh ) , 8.708.47 ( m , 6h ) , 7.90 ( d , j = 7.8 hz , 2h ) , 7.637.18 ( m , 8h ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 162.0 , 160.1 , 149.6 , 148.5 , 144.0 , 143.7 , 137.0 , 123.2 , 134.0 , 133.6 , 120.0 , 129.2 , 129.1 , 125.2 , 123.2 , 122.8 , 119.8 , 20.6 . h nmr ( 300 mhz , cdcl3 ) : 9.13 ( bs , 1h , nh ) , 8.778.66 ( m , 3h ) , 7.717.16 ( m , 11h ) , 4.82 ( s , 2h , ch2 ) , 2.36 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 161.3 , 150.8 , 144.6 , 143.9 , 136.9 , 132.9 , 130.5 , 129.7 , 129.4 , 128.7 , 128.6 , 128.0 , 121.6 , 120.3 , 44.3 , 21.1 . hrms calcd for c25h22n5o2 [ m+h ] : 424.1768 ; found : 424.1768 . yield : 73% ; h nmr ( 300 mhz , cdcl3 ) : 8.94 ( bs , 1h , nh ) , 8.688.62 ( m , 4h ) , 8.49 ( bs , 1h , nh ) , 8.37 ( d , j = 8.4 hz , 1h ) , 7.63 ( d , j = 8.4 hz , 2h ) , 7.487.14 ( m , 6h ) , 2.42 ( s , 3h , ch3 ) , 2.37 ( s , 3h , ch3 ) . c nmr ( 75 mhz , cdcl3 ) : 161.3 , 150.5 , 146.4 , 144.5 , 144.1 , 134.8 , 131.5 , 130.4 , 129.8 , 124.5 , 123.2 , 120.3 , 21.1 . hrms calcd for c25h22n5o2 [ m+h ] : 424.1768 ; found : 424.1770 . green monkey kidney cells [ vero - b cells as obtained from the european collection of cell cultures ( ecacc ) ] were grown in minimum essential medium ( mem ; gibco , merelbeke , belgium ) supplemented with 10% fetal calf serum ( fcs ) , 1% l - glutamine and 1% sodium bicarbonate . vero - b cells were seeded at a density 7 10 cells / well in 100 l assay medium and allowed to adhere overnight . antiviral assays were performed in medium supplemented with 2% fcs , 1% l - glutamine and 1% sodium bicarbonate . after washing cells twice with 2% fcs medium , serial compound dilutions ( 1:2 ) were added to each well ( starting concentration 100 g / ml ) , following by adding 100 l of 2% of phosphate buffered saline ( pbs ) culture medium containing 100 l 50% cell culture infectious doses ( i.e. , ccid50 ) of virus . after 7 days of incubation , 2% fcs culture medium was discarded and cells were fixed with ethanol and stained with 1% methylene blue , and ec50 and cc50 were determinate visually . the 50% effective concentration ( ec50 ) , which is defined as the compound concentration that is required to inhibit the virus - induced cytopathogenic effect ( cpe ) by 50% , and 50% cytotoxic concentration ( cc50 ) , which is defined as the compound concentration that is required to inhibit the cell growth by 50% .

the results of a high - throughput screening assay using the dengue virus-2 replicon showed that the imidazole 4,5-dicarboxamide ( i45dc ) derivative ( 15a ) has a high dengue virus inhibitory activity . based on 15a as a lead compound , a novel class of both disubstituted i45dcs and the resembling pyrazine 2,3-dicarboxamides ( p23dcs ) were synthesized . here , we report on their in vitro inhibitory activity against dengue virus ( denv ) and yellow fever virus ( yfv ) . some of these first generation compounds have shown activity against both viruses in the micromolar range . within this series , compound 15b was observed to display the highest antiviral potency against yfv with an ec50 = 1.85 m . in addition , compounds 20a and 20b both potently inhibited replication of denv ( ec50 = 0.93 m ) in vero cells .

Introduction Results and discussion Conclusion Materials and methods

PMC2288681

dna - functionalized gold nanoparticles are an interesting system with applications ranging from biological sensors to the construction of self - assembled materials . experiments are based on attaching single - stranded dna molecules via thiol - gold bonds to the surface of au nanoparticles and a subsequent self - assembly process of these conjugates by making use of base pairing of complementary dna molecules [ 15 ] . for example , by employing au - dna conjugates , several groups have developed schemes to detect target dna sequences and to assemble nanoparticles into macroscopic materials [ 7 , 8 ] . dna - functionalized au nanoparticles are the building blocks for the above - mentioned experiments . therefore , it is of great interest to investigate the properties of these conjugates in detail . due to the high affinity of thiol groups to gold surfaces , thiol - modified dna molecules can be directly bound to the surface of citrate- or phosphine - stabilized au nanoparticles [ 9 , 10 ] . although commonly a random number of dna molecules are attached per au nanoparticle , also particles with an exactly defined number of one , two , or three attached dna molecules per nanoparticles can be obtained [ 1115 ] . certainly , several parameters have significant influence on the properties of au - dna conjugates , such as coverage of the au surface with dna , configuration of the attached dna molecules , and hybridization efficiency of dna attached to au surfaces . the degree of dna coverage will influence the dna conformation , which , in turn , will affect the hybridization efficiency . a body of experiments investigating these parameters has been reported for dna attached to flat au surfaces using different techniques such as atomic force microscopy ( afm ) [ 1618 ] , surface plasmon resonance ( spr ) spectroscopy [ 1921 ] , radioisotopic techniques [ 22 , 23 ] , ellipsometry , and x - ray photoelectron spectroscopy ( xps ) . these experiments allow for a detailed picture of dna bound to planar gold surfaces and the results have clarified the binding mechanism , the surface coverage , the hybridization efficiency , and the role of nonspecific adsorption , all in dependence of the length of the dna . since the effect of surface curvature has to be taken into account , the results obtained for planar au surfaces may be transferred to spherical au nanoparticles only under certain restrictions . the surface coverage of au nanoparticles with dna has been investigated using the displacement of fluorescence - labeled dna molecules with mercaptoethanol and by gel electrophoresis . also , the conformation of bound dna [ 27 , 28 ] , hybridization , and the role of nonspecific adsorption [ 26 , 28 , 30 ] have been investigated for au nanoparticles . in this report , we present a detailed study of electrophoretic mobility of au - dna conjugates . with this study , we want to determine the possibilities and limitations of this technique . besides our own previous work [ 27 , 31 ] , also other groups [ 28 , 32 , 33 ] have recently reported about the possibility to extract effective diameters for bioconjugated colloidal nanoparticles from electrophoretic mobilities . the aim of this study is , in particular , to investigate the limitations of this analysis . citrate - coated gold nanoparticles of 5 , 10 , and 20 nm diameter were purchased from bbi / ted pella ( redding , calif , usa ) . in order to improve their stability in buffer solution , the adsorbed citrate molecules were replaced by a phosphine ( bis(p - sulfonatophenil)phenylphosphine dehydrate , dipotassium salt ) . the concentration of the au nanoparticles was determined by uv / vis spectroscopy by using the molecular extinction coefficient of their absorption at the plasmon peak . thiol- and cy5-modified and unmodified single - stranded dna were purchased from idt ( coralville , iowa , usa ) or metabion ( mnchen , germany ) . all sequences can be found in the supplementary material ( available online at doi : 10.1155/2007/26796 ) . the concentration of the dna was determined by uv / vis spectroscopy by using the molecular extinctions coefficient of their absorption at 260 nm . the thiol - modified and plain dna were added to the phosphine - coated au nanoparticles at ph = 7.3 , c ( nacl ) = 50 mm , and samples were incubated for some hours up to several days [ 11 , 27 ] . generally , in such experiments , dna is always added in large excess , thus that the number of attached molecules is related but not fully controlled by the stoichiometry of dna : au - np because of the rather low binding yield . the resulting au - dna conjugates were loaded on 0.5%6% agarose gels ( agarose : gibco brl , number 15510 - 027 ; 0.5 tbe buffer , ph 9 ) and run for one hour at 100 v [ 11 , 27 ] . ( since 6% gels can be inhomogeneous due to their high viscosity , the data obtained with these gels have to be interpreted with care . ) as reference , always unconjugated au nanoparticles of the same diameter were run on the same gel . in addition , gels with unconjugated au nanoparticles of different diameter and free dna of different length were run . the bands of the plain and dna - conjugated au nanoparticles were directly visible by the red color of the au colloid and the free dna was visualized by an attached fluorescence label ( fluorescein , cy3 , or cy5 ) . the bands of the gels were photographed using a digital camera system ( eagle eye iii , stratagene ) . the mobility of each sample was determined by measuring the position of each band referring to the start position where the samples had been loaded . this resulted in a comprehensive set of data which relates the mobility of au - dna conjugates to the diameter of the au particles , where the relation between the amount and the length of the attached dna , nonspecific versus specific attachment via thiol - gold bonds , and the gel percentage was studied . since mobility is not an illustrative quantity , we have attempted to convert the mobilities of au - dna conjugates in effective diameters . the evaluation of the gels in which plain au - nanoparticles of known diameter were run yielded a calibration curve in which the mobility is plotted versus the diameter . by using this calibration curve , the mobility of the au - dna conjugates could be directly converted into effective diameters . alternatively , the mobility of au - dna conjugates at different agarose concentrations was used to obtain ferguson plots and fits of the ferguson plots yielded the retardation coefficients . first , ferguson plots were made for plain au - nanoparticles of known diameter and a calibration curve in which the retardation coefficients were plotted versus the particle diameter was obtained . by using this calibration curve , the effective diameters of au - dna conjugates could be derived from the retardation coefficients derived from the ferguson plots of the au - dna conjugates . we have also quantified the maximum number of dna molecules that can be attached per gold nanoparticle for particles with 5 nm and 10 nm diameter and single - stranded dna with 8 and 43 bases . for this purpose , single - stranded dna that had been modified with a thiol group on the 3 ' and a cy5 dye on the 5 ' end has been attached via formation of thiol - au bonds to the surface of au particles . dna was added in different dna to au ratios and the conjugates were run on an agarose gel . the more dna bound per au nanoparticle , the more the band of this conjugate was retarded on the gel . at a certain amount of added dna , the retardation of the band of the conjugates did not further increase , which indicates that the au surface is fully saturated with dna . the bands were extracted from the gel by cutting out the agarose piece that contained the band and immersing it into 0.5 tbe buffer solution . after two days , the au - dna conjugates had diffused out of the gel into the buffer . the extraction procedure ensures that all dna is really attached to the au particles , since free dna migrates in a much faster band . uv / vis spectra were recorded of the extracted au - dna conjugates . for each of the conjugates , the dna concentration was determined by the cy5 absorption and the au concentration was determined by the absorption at the plasmon peak and from both concentrations the number of attached dna molecules per particles was derived . as we quantified the number of attached cy5 molecules only with absorption and not with fluorescence measurements , the effect that the fluorescence of cy5 close to au surfaces is quenched all methods and additional experiments can be found in detail in the supplementary material . the attachment of dna to au nanoparticles can be clearly observed by gel electrophoresis [ 9 , 11 , 13 , 2628 , 3638 ] . the mobility of particles on the gel depends on two factors : size and charge . the bigger the size , the slower and the higher the charge , the faster particles will migrate . in the case of negatively charged au particles ( e.g. , with citrate or phosphine molecules adsorbed to the particles ) , the attachment of negatively charged dna molecules causes in first place an increase of size that can be seen as a retardation of the band of the gel . if the change in charge dominated , then the mobility of the au particles should be increased ( addition of negative charge ) or drastically decreased ( addition of positive charge ) up to change in the direction of migration . although this effect has been observed for different systems [ 31 , 39 ] , it has not been observed for the au - dna conjugates used in this study . upon attachment of dna , therefore , in agreement with previous reports , we assume throughout this manuscript that attachment of dna to au nanoparticles in first order increases the effective diameter of the conjugates which can be directly seen in the retardation of the band of the conjugates in gel electrophoresis experiments [ 9 , 11 , 2628 , 3638 ] . one aim of this study was to obtain calibration curves in which measured electrophoretic mobilities m can be related to effective diameters deff . by running phosphine - stabilized au particles of known diameter ( the overall diameter of phosphine - coated au np was assumed as the core diameter plus two times 0.5 nm for the thickness of the phosphine layer and the smallest nanoparticle size used for calibration was 6 nm ) on gels , by measuring their mobility , by fitting the data empirically with an exponential function , and by using the inverse of the fit function , we obtained a function in which the effective diameter of au particles and au - dna conjugates can be directly calculated from their electrophoretic mobility : ( 1)deff(m)=ty*ln((m / m10 nm , y)/ay)+6 nm . the parameters for y = 0.5% , 1% , 2% , 3% , 4% , 5% , and 6% agarose gels are enlisted in table 1 . in order to enhance the accuracy by making relative instead of absolute measurements , we always normalized the mobilities m to the mobilities m10 nm , y of plain phosphine - stabilized au particles of 10 nm core diameter on the same gel . therefore , although the primary data of all electrophoresis measurements are electrophoretic mobilities , we are discussing the experimental results in terms of effective diameters . the diameters have been obtained with the above - described formula from the mobility data . since obviously the effective diameter of au - dna conjugates is a fixed physical property , it should not depend on the form of measurement and analysis . we , therefore , compared the effective diameters derived from 1% , 2% , 3% gels via the respective mobility - diameter calibration curves and from ferguson plots . for the ferguson plots , the determined effective diameters for au - dna conjugates for au particles saturated with dna and for au particles with only few dna strands attached per particle are plotted in figures 1 and 2 for dna of different length . in all cases , regardless the length of the dna , whether dna was attached by specific thiol - gold linkage or by nonspecific adsorption , or whether only a few or a many as possible dna molecules were bound per au nanoparticles , the effective diameters derived with the mobility - diameter calibration curves are different for different gel percentages . though most of the times the effective diameters derived from gels with higher percentage were found to be larger than the ones obtained from gels with lower percentage , also the opposite effect was observed within the experimental error bars ( see , e.g. , figure 2 ) . the effective diameters derived from ferguson plots were always smaller than the ones derived from the mobility - diameter calibration curves . if always the effective diameters derived from the gels of higher percentage were smaller than the one derived from gels with lower percentage , one could have argued that the soft dna shell around the rigid au cores would be squeezed or compressed more while migrating through the gel of higher agarose concentration , which would lead to smaller effective diameters . however , since no clear correlation between the gel concentration and the derived effective diameters was observed , we have to consider the difference between the effective diameters that have been obtained from gels of different concentrations as error bars . the bigger the au particles become due to attachment of dna , the bigger the error in deriving their effective diameter from electrophoretic mobilities becomes . for example , according to figure 1 , the effective diameters of 10 nm au particles saturated with 100 bases dna that is specifically linked via thiol - au bonds are 66.3 nm , 69.5 nm , and 58.5 nm as determined from 1% , 2% , and 3% gels . we believe that from these data we can assume that the effective diameter of these conjugates is around 60 nm with an error bar of around 10 nm . from these and additional similar data ( not shown ) , we conclude that deriving absolute effective diameters from electrophoretic mobilities via mobility - diameter calibration curves is possible only under certain restrictions . it is not sufficient to extract the data just from gels of one percentage . only by using gels of different percentage an average value for the effective diameter and an estimate about the error can be obtained . part of this limitation might be due to our principal assumption that in the case of phosphine - stabilized au particles conjugated with dna , the electrophoretic mobility is in first order only determined by the size of the conjugates . for other systems in which charge effects certainly will play a more important role , it might be even impossible to derive effective diameters from electrophoretic mobilities with the here - reported mobility - diameter calibration curves . it also has to be pointed out that the possible application of the here - reported calibration curves is limited to relatively rigid objects similar in nature to au nanoparticles . as these objects were used in first order to obtain the experimental data on which the calibration functions are based , the calibration functions certainly will not describe the diameters of soft objects , such as dna , very well . a likely explanation for the deviation in the effective diameters obtained for the dna - au conjugates with the calibration functions for the gels of different percentage can be seen in the fact that the calibration functions are directly only applicable for au particle - like rigid objects . attaching soft objects as dna to the au particle surface changes their electrophoretic behavior so that the calibration curves can be only applied in a restricted way . we have also evaluated the possibility to obtain effective diameters of au - dna conjugates via ferguson plots , as had already suggested by the group of hamad - schifferli . from figures 1 and 2 , it is evident that the effective diameters obtained from ferguson plots are always significantly smaller than the ones obtained from mobility - diameter calibration curves . it has to be pointed out that both evaluation methods are based on the same set of experimentally obtained mobilities . in a classical ferguson plot , for example for free dna , however , in the case of au and au - dna conjugates , this linearity holds no longer true , in particular for gels of higher percentage . we , therefore , had to restrict our analysis to gels from 1% to 3% although in some cases data for 4% to 6% had also been available . though theories for nonlinear , convex ferguson plots exist [ 40 , 41 ] , we did not try to apply them here . due to the significant deviation from the data obtained with the ferguson plots to the data obtained with mobility - diameter calibration curves and due to the above - mentioned limitations , we conclude that the linear ferguson analysis is less suited to obtain absolute effective diameters . however , relative increases in size due to binding of molecules can be observed with sufficient resolution with ferguson analysis . our data clearly indicate that there is also nonspecific adsorption of dna to the surface of au particles in case the particles are exposed to many dna molecules , see figure 1 . it is important to point out that in figure 1 , the data of au particles that have been exposed to as much dna as possible and that are , therefore , saturated with dna are described . this is different from the case in which the au particles are exposed to only to a few strands of dna as in figure 2 , where no nonspecific adsorption could be observed , as already reported by zanchet et al . . nonspecific adsorption of dna to au particles is significantly lower compared to specific thiol - au bond - mediated attachment and thus can only be observed in case of exposure of the particles to very high dna concentrations . although the absolute numbers derived for effective diameters for au - dna conjugates are afflicted with significant error bars as described above , these data nevertheless contain valuable information about the binding of dna to au particles . any attachment of dna leads to an increase in the effective diameter , dependent on the nature of attachment , the amount of bound dna , and the length of each dna molecule , see figure 1 . with very simple models , we can assume that dna attached to the surface of au particles can adopt two basic types of conformation . in the first case , the confirmation of dna is not effected by the presence of the au particles and it will form a random coil . in the second case , dna has to compete for the binding places at the gold surface and thus , in order to bind as many dna molecules per area as possible , the dna has to be stretched . in figure 1 , the effective diameters for the different models ( randomly coiled dna , fully stretched dna , and dna that is stretched for the first 30 bases and randomly coiled for the rest of the bases ) are plotted versus the dna length for au particles that are saturated with dna . clearly , thiol - gold - bond specific attachment can be distinguished from nonspecific adsorption of dna . similar observations have been reported also before by sandstrm et al . [ 26 , 37 ] . first , the increase in the effective diameter tells that also dna without thiol modification can be adsorbed to the surface of phosphine - stabilized au nanoparticles . second , a comparison with the effective diameters of the theoretical models clearly proves that nonspecifically adsorbed dna does not exist in a stretched configuration perpendicular to the au surface . the data rather indicate that even when the particle surface is saturated with nonspecifically attached dna , only parts of the dna molecules will be randomly coiled , as the experimentally obtained effective diameters are smaller than the diameter of conjugates in which the adsorbed dna is randomly coiled . from this , one can conclude that due to nonspecific au - dna interaction , the adsorbed dna is at least partly wrapped around the surface of the au particles , which is in agreement with other studies . in case of au surfaces saturated with thiol - modified dna , the effective diameters are significantly bigger compared to nonspecifically adsorbed dna , see figure 1 . by comparison with basic models , we conclude in agreement to our previous study that specifically bound dna adopts a stretched configuration so that as many dna molecules as possible can bind to the au surface . due to the spherical geometry , dna longer than around 30 bases only needs to be stretched due to this space limitation within around the first 30 bases , whereas the parts of the dna molecules further away from the au particle are not affected by space limitation and thus can be randomly coiled . these results again show the possibilities and limitations of the here - described method . though it is complicated to derive accurate absolute effective diameters of au - dna conjugates , the binding of dna molecules can be clearly seen as an increase in the effective diameters and a comparison with theoretical models can give indications about the conformation of the attached dna . these types of binding assays via gel electrophoresis are an attractive complementary method compared to other techniques , such as light scattering . presumably a combination of gel electrophoresis , light scattering , and zeta potential measurements of identical samples would give the most accurate analysis about au - dna conjugates . it remains to note that although electrophoresis of free dna is well studied both experimentally and theoretically , the case of au - dna conjugates is more complex because several properties ( total charge , charge density , and elasticity ) are not constant but depend all at the same time on the binding of dna to the au nanoparticles . a theoretical model for gel electrophoresis of such conjugates would be helpful for data analysis . when organic fluorophores are attached to au - dna conjugates at the free end of the dna , which is pointing towards solution , then energy transfer between the fluorophore and the au nanoparticle can be observed . this effect can be , for example , employed for dna sensors . since energy transfer depends on the distance between the organic fluorophore and the au surface [ 35 , 47 ] , certainly the configuration of the bound fluorophore - modified dna is important for this process . in case of nonspecific adsorption of the fluorophore to the au surface , the distance between the fluorophore and the au would be much smaller than for the case in which the dna is linked with its thiol - modified end , see figure 3 . in this study , we have shown that the attachment of cy5 to the free end of thiol - modified dna does not change the effective diameter in the case of au particles saturated with dna , see figure 3 . these results demonstrate that the direct adsorption of cy5 to the au surface is much less probable than the formation of thiol - au bonds and that , therefore , the dye points towards the solution . the number of bound dna molecules per au particle has already been determined with several methods [ 25 , 26 , 48 ] . in comparison to methods in which the number of dna molecules is quantified by the fluorescence of attached fluorophores , the counting of dna via absorption measurements ( as reported in this study ) is not affected by photobleaching and quenching effects . extracting the au - dna conjugates from the gel also helps that no unbound excess dna is present in the solution , as it still might be possible in the case of purification with filter membranes . the results of this study are summarized in figure 4 and are in the same range as the results obtained by other groups [ 25 , 26 , 48 ] though our determined dna densities are rather lower than the ones determined by other groups . this might be due to the fact that the phosphine stabilization is harder to be displaced by dna than citrate stabilization and in particular due to the fact that our incubation was performed at lower nacl concentrations . in our measurements , we could not find any effect of the different curvature between 5 nm and 10 nm gold particles on the density of attached dna molecules . this can be understood as the surface curvature difference between both types of particles is not very high and dna attachment to both types of particles was done under the same buffer conditions . recently , qin and yung have instead demonstrated that the most relevant parameter for the maximum number of attached dna molecules per particle is the salt concentration under which the attachment was performed . high salt concentrations reduce electrostatic repulsion und thus allow for higher dna surface densities . as already reported in earlier publications , gel electrophoresis allows for a separation of au - dna conjugates with 0 , 1 , 2 , dna molecules attached per particle [ 9 , 11 ] . in figures 2 and 5 , the effective diameters of such conjugates as determined from their electrophoretic mobilities are presented . the dependence of dna length and au core diameter on the effective diameter is as expected . the longer the dna , the more the effective diameter of au - dna conjugates upon which attachment of another dna molecule to one gold particle is increased ( see figure 2 ) . the more long dna strands are attached per individual gold particle , the fewer the effective diameter of the au - dna conjugated depends on the initial diameter of the au core ( see figure 5 ) . although no simple model for au - dna conjugates is available that could predict the exact mobility in gel electrophoresis , the bands of particles with a defined number of dna strands can be identified with their structure by relative ( qualitative ) comparison and control experiments that include hybridization . so far , we are not aware of another separation technique ( such as hplc ) that can resolve au particles with an individual number of attached dna molecules as it is possible with gel electrophoresis . the concept of separating conjugates of particles with a discrete number of attached molecules by gel electrophoresis could be also be generalized and used besides for au - dna conjugates for other systems . because of their defined composition , we think that such conjugates of particles with a defined number of linked molecules are very interesting model systems and several applications have been already demonstrated [ 50 , 51 ] .

gold - dna conjugates were investigated in detail by a comprehensive gel electrophoresis study based on 1200 gels . a controlled number of single - stranded dna of different length was attached specifically via thiol - au bonds to phosphine - stabilized colloidal gold nanoparticles . alternatively , the surface of the gold particles was saturated with single stranded dna of different length either specifically via thiol - au bonds or by nonspecific adsorption . from the experimentally determined electrophoretic mobilities , estimates for the effective diameters of the gold - dna conjugates were derived by applying two different data treatment approaches . the first method is based on making a calibration curve for the relation between effective diameters and mobilities with gold nanoparticles of known diameter . the second method is based on ferguson analysis which uses gold nanoparticles of known diameter as reference database . our study shows that effective diameters derived from gel electrophoresis measurements are affected with a high error bar as the determined values strongly depend on the method of evaluation , though relative changes in size upon binding of molecules can be detected with high precision . furthermore , in this study , the specific attachment of dna via gold - thiol bonds to au nanoparticles is compared to nonspecific adsorption of dna . also , the maximum number of dna molecules that can be bound per particle was determined .

1. INTRODUCTION 2. MATERIALS AND METHODS 3. RESULTS AND DISCUSSION

PMC3958141

docking is a well - established computational technique often used at the early stages of structure - based drug discovery . in general , the purpose of a docking experiment is to predict the optimal binding geometry ( pose ) of a ligand ( typically a small organic molecule ) with respect to a receptor ( typically a protein drug target ) , and to provide an estimate of the interaction affinity relative to other ligands . dock is one such program designed for binding pose prediction and , when used in a virtual screening capacity to rank - order large databases of candidate molecules , is a powerful approach for lead discovery . numerous dock successes have been reported ; recent examples include identification of leads targeting ctx - m -lactamase , 2-adrenergic receptor , xpa protein , and the riboswitch of mrna , among others . examples of successful virtual screens from our laboratory employing the most recent version of dock ( version 6 ) include the drug targets hivgp41 and fatty acid binding protein . other related programs , including autodock , autodock vina , gold , glide , surflex , and flexx , are widely used for similar goals and have documented analogous successes ( reviewed in yuriev et al . ) . root - mean - square deviation ( rmsd ) , a measure of intermolecular differences in position and conformation , has broad applications in computational biology , including testing the efficacy of docking programs . for example , in a typical docking validation experiment , a cocrystallized ligand is removed from its binding site , then multiple candidate binding poses are predicted using a specific protocol . the positive - control experiment is successful if the heavy - atom rmsd of the top - ranked pose is within a certain tolerance from the original ligand position ; a typical cutoff for success is 2.0 . however , if a molecule contains symmetric functional groups or is itself symmetric , inflated rmsd values and a false negative result can occur . as an example , docking 1,2-dichlorobenzene to t4 lysozyme ( pdb code 2oty ) can yield chemically equivalent binding poses , yet different rmsd values ( figure 1 ) . in one scenario , the pose rmsd is less than 2.0 from the crystallographic reference and is considered a docking success . in the second scenario , the pose is inverted over its axis of symmetry , yielding an rmsd greater than 2.0 , which is reported as a docking failure . dock6 was recently reported to successfully redock between 63.8% ( over 780 systems ) and 66.3% ( over 147 systems ) of known crystallographic ligand receptor complexes in a series of flexible ligand docking experiments using a standard energy - based scoring function . a subset of the failures from these experiments could be attributed to molecular symmetry in the ligand . when symmetry is accounted for , an increase in success of approximately 48% these trends demonstrate that neglecting to account for symmetry in ligands can negatively impact the accurate evaluation of new docking programs , protocols , search methods , or scoring functions . docked poses , shown in orange , are inverted approximately 180 about the axis of symmetry . , there remains a surprising lack of published details regarding specific functions used for symmetry correction , as was recently noted by brozell et al . a notable exception is the publication describing autodock vina , which reports the exact procedure used to compute what the authors refer to as the minimum - distance rmsd . however , while functionally simple to implement , minimum - distance rmsd in some cases may not enforce a one - to - once atom correspondence . under these circumstances , some atoms could be used multiple times for the rmsd calculation while others could be neglected , which may not be desirable ( see theoretical methods ) . as an alternative approach , our group has implemented a symmetry - correction strategy into the dock6 program based on the method described by kuhn and later by munkres known as the hungarian algorithm . a similar strategy was recently reported by helmich et al . to find the best overlap in alignment between two conformations of the same molecule , and by ellingson et al . to efficiently maximize the number of atom superpositions in an overlay of protein binding sites . in this report , we demonstrate that our implementation of the hungarian algorithm effectively corrects for symmetry in molecular docking which provides the means to assess the impact of various algorithmic or protocol changes as the dock program continues to evolve . further , we discuss an extension of the algorithm for adding chemical diversity to a pool of molecules that are assembled in a de novo design version of dock currently under development , enabling improved sampling of diverse chemotypes . additional applications presented include rank - ordering compounds relative to a known reference to aid virtual screening and computing symmetry - corrected rmsds to analyze molecular dynamics simulations . the standard pairwise rmsd function , as it is most commonly used in structural biology , quantifies the difference between two poses of the same molecule . in this context , differences between poses can include rigid geometric rotations and translations within the six standard degrees of freedom ( three rotational and three translational ) , plus any changes in internal degrees of freedom ( such as dihedral rotations ) . the standard rmsd function ( rmsdstd ) is defined as follows:1where molecule poses a and b are sets comprising individual atoms : a = { a1,a2 , ... , an } and b = { b1 , b2 , ... , bn}. atoms ai and bi can be interpreted as cartesian coordinates or positional vectors : for example , ai = { xa , i , ya , i , za , i}. the total number of heavy atoms in the molecule is n. historically , this function is dependent on a predefined one - to - one correspondence between atoms in two poses ( a and b ) of the same molecule . for practical purposes , this is typically achieved by matching atom indices or evaluating the sets of atoms in the same order . in the case of highly symmetric molecules , however , it would be advantageous to impose a dynamic atom - matching algorithm prior to the standard rmsd calculation that is not subject to the constraints of arbitrary atom numbers or orders , such that physically indistinguishable atoms would be matched . in an attempt to overcome this limitation , trott and olson recently described a modified function termed minimum - distance rmsd ( rmsdmin ) , computed as follows:2where3 in this method , atoms ai from reference pose a are iteratively compared to all atoms bj from pose b that are of the same element type . the minimum distance identified defines the atom correspondence , which is subsequently used in the root - mean - square calculation . the same procedure is repeated using pose b as the reference , and the maximum of the two calculations is reported as the rmsdmin . however , as this function does not enforce a one - to - one correspondence , some atoms may be used more than once in the calculation while others that are far removed in coordinate space have the potential to be neglected . therefore , while this method is effective , it can under - represent the true rmsd . in order to account for all atoms in each pose a and b , the minimum - distance rmsd could be modified to enforce a one - to - one correspondence through removal of atom pairs after they have been matched via following the steps : ( 1 ) for each i , compute the minimum distance : minj||ai bj|| , ( 2 ) remove ai and bj from atom sets a and b , respectively , and ( 3 ) repeat steps 12 until no atoms remain to be matched . once the complete atom correspondence is determined , the rmsd would be computed similar to eq 3 . however , while this modified approach would ensure that no atoms are neglected from the final rmsd calculation , it is dependent on the order in which atoms are evaluated and may give a nonoptimal solution . as an alternative approach , the optimal one - to - one correspondence between sets of atoms can be solved using the hungarian algorithm , a method for solving the minimum assignment problem . in this approach , every atom from pose a will be matched to exactly one atom from pose b in such a way that minimizes the sum of distances between all atom pairs , and no outlying atoms will be neglected from the rmsd calculation . the optimal - correspondence rmsd ( rmsdcor ) , termed here the symmetry - corrected rmsd , can be defined as follows:4where atom ai is matched uniquely to an atom bj that was predetermined by solving the optimal correspondence corj||ai bj|| . briefly described , the hungarian algorithm solves this correspondence in four steps : ( 1 ) form a cost matrix m where each matrix element mij is the squared norm of atom positional vectors ai and bj . subtract the lowest value in each row from all elements in the same row and the lowest value in each column from all elements in the same column . ( 2 ) find the minimum number of horizontal and vertical lines that can cover all zeroes in the matrix . if the number of lines is equal to the size of the matrix in one dimension , a set of independent zero - values exists , and the positions of these zeroes represent the optimal - correspondence solution ( 3 ) determine the smallest matrix element that is not covered with a line . subtract that value from all uncovered matrix elements and add that same value to all matrix elements that are covered by two lines . ( 4 ) repeat steps 23 until the matrix is solved as described in step 2 . each iteration of steps 23 constitutes a cycle of the algorithm . a more thorough description and proof are documented by munkres . a very fast , o(n ) version of the hungarian algorithm was implemented into the source code of dock6 . disjoint distance matrices ( termed submatrices ) are assembled for each atom type ( using tripos sybyl conventions ) in a given molecule , thus only atoms of the same type can correspond to one another in the rmsd calculation ( see figure 2 ) . all tests were performed on a dell poweredge c6100 linux cluster consisting of intel x5660 2.8 ghz hex - core nehalem - based processors . in this example , a theoretical molecule that contains only four sybyl atom types ( o.3 , f , c.3 , and n.3 ) is described . the search space was reduced from all possible atom comparisons ( left ) to comparisons between only atoms of the same atom type ( right ) . the amount of calculation required , represented by blue shading , is significantly decreased by dividing the molecule into subsets , or submatrices . pose - reproduction docking experiments were performed over a large test set using the flexible ligand protocol described by mukherjee et al . the test set used is an expanded version of the sb2010 test set ( n = 780 ) recently increased to include 1043 receptor ligand complexes , available for download as sb2012 at www.rizzolab.org . key aspects of the test set preparation and flexible ligand docking protocol include the following : ligands were protonated within the program moe , then semiempirical am1-bcc charges were added with antechamber , part of the ambertools package . receptors were protonated and assigned force field parameters with tleap ( ambertools ) , then the receptor structures were subjected to a short energy minimization using sander ( amber11 ) with strong restraints on the heavy atoms . following standard dock6 practices , the receptor surface was generated with dms , spheres were generated using the utility sphgen , and a box was defined that surrounded the spheres plus an 8.0 margin in all directions . a docking grid was generated within the boundaries of the box at 0.3 resolution with 69 van der waals exponents and a distance - dependent dielectric of = 4r . during docking , a maximum of 1000 anchor orientations ligand positions were minimized during growth with a simplex minimizer for a maximum of 500 iterations or until the change in energy score between steps was less than 1.0 kcal / mol . a maximum of 5000 fully grown conformers were kept for each ligand , following best - first clustering with a 2.0 standard rmsd threshold . the resulting ensembles of candidate poses ( 88 853 total poses ; average 85.2 poses per system ) were evaluated using both the standard ( eq 1 ) and symmetry - corrected ( eq 4 ) rmsd algorithms described above . all rmsd values reported were measured between the docked pose and the crystallographic position of the same ligand . rmsd values are computed using heavy - atoms only hydrogen atom positions were not considered . as an alternative application of the hungarian algorithm , we have adapted the code to enable clustering and pruning between molecules of differing chemical composition in order to aid in de novo design . depending on several factors , the number of candidate molecules during de novo assembly can quickly expand exponentially , thus rigorous pruning methods are required to keep the search space tractable . to evaluate the impact of the new pruning heuristic , experiments to reconstruct known molecules and to construct diverse ensembles of molecules were performed using a de novo version of dock currently under development ( further discussed in balius et al . ) . prior to the de novo experiments , two different groups of molecule fragment libraries were prepared . first , all ligands from the sb2012 test set with exactly seven rotatable bonds ( n = 103 ) were independently disassembled on those bonds into 103 small , restricted fragment libraries . second , a subset of the zinc database consisting of approximately 1 10 representative drug - like molecules was used to form a larger , generic fragment library . new molecules were then assembled de novo in the binding sites of each receptor from the sb2012 subset using either ( 1 ) the smaller , restricted fragment libraries associated with each system or ( 2 ) the larger , generic library from zinc seeded with fragments from the restricted libraries . following each layer of assembly , molecule ensembles were rank - ordered by the value of their dock grid energy score , then pruned using a best - first clustering method and the adaptation of the hungarian algorithm . the criteria for pruning were as follows : all possible atom matches ( using sybyl conventions ) between the two molecules were identified , the optimal - correspondence of those matches was determined , and the symmetry - corrected rmsd for matched atoms was computed ( hereafter referred to as rmsd - matched ) . in addition , the atoms that remained unmatched in either molecule were enumerated ( hereafter referred to as # -unmatched ) . two molecules were deemed to be sufficiently similar in cartesian space and chemical space if the rmsd - matched term was less than a user - defined cutoff ( typically 2.0 ) , and the # -unmatched term was less than a second user - defined cutoff ( typically 010 atoms ) . under these conditions , the worse - scoring molecule would be pruned . in practice , as the threshold for number of unmatched atoms increases , molecules would be pruned more frequently . for clarity tanimoto coefficients were computed using a fingerprinting method implemented into dock6 , inspired by the molprint 2d algorithm described by bender et al . comparison between a gray reference molecule ( same in all four panels ) and four example molecules constructed by de novo design with : ( a ) slight differences in functional group positions , ( b ) larger differences in functional group positions , ( c ) slight differences in functional group composition , and ( d ) larger differences in functional group composition . in total , 88 853 docked poses derived from the sb2012 test set were compared against 1043 crystallographic reference conformations using the standard and symmetry - corrected rmsd functions . the present implementation of the hungarian algorithm was made significantly more efficient through a process where submatrices are prepared containing only atoms of the same sybyl atom type for matching ( see figure 2 ) . in terms of the chemistry of matching atoms , it follows that only atoms of an identical topology should be matched to correctly account for molecular symmetry . in order to verify that the hungarian algorithm could successfully solve each minimum assignment problem in the data set , we measured the number of algorithm cycles required for each calculation . the 88 853 poses were divided into 675 056 submatrices based on atom type ( average 7.60 submatrices , or atom types , per molecule ) and the minimum assignment was determined . scatter plot of the number of cycles of the hungarian algorithm ( y - axis ) required to solve the minimum assignment problem for matrices of a given size ( x - axis ) . each red point represents one submatrix ( n = 675 056 ) . the maximum number of algorithm cycles required to solve the most difficult matrix was 265 cycles , which occurred in a matrix of 33 33 elements . in addition , the largest matrix evaluated in the data set was 35 35 elements , derived from a molecule with 138 total atoms , of which 35 atoms were the same sybyl atom type ( sp3 carbon , pdb code 1kqy ) . on average , the complexity of solving the minimum assignment problem , as measured by the number of algorithm cycles required , increases with increasing number of matrix elements . conceptually this is consistent with increasing opportunity for alternative atom correspondence among larger ligands . figure 4 also demonstrates that all distance matrices from a very large data set were solvable , exhibiting the robustness of the algorithm . finally , it is noteworthy that rescoring all 88 853 poses ( 675 056 total submatrices ) required 77 981 ms total of computer time . this value factors to an average of 0.88 ms per pose or 0.12 ms per submatrix to solve the minimum assignment problem . submillisecond time scales for evaluating the symmetry - corrected rmsd are negligibly small when compared to an average on - the - fly flexible ligand docking experiment in the program dock6 , which under the present conditions required approximately 4.9 min per ligand . this millisecond time scale becomes an important consideration when , as discussed below , the algorithm is adapted to enable pairwise comparisons for de novo design . by design of the algorithm , the symmetry - corrected rmsd can only be less than or equal to the standard rmsd . that is , the atom correspondence will not change from the standard correspondence if any change increases the sum of distances between all atom pairs . a heat map of rmsd comparisons for all 88 853 poses is shown in figure 5 . data here are grouped in 0.2 bins , red indicates areas of high population , green indicates areas of low population , and white areas are unpopulated . the heat map can be divided into four quadrants : poses in quadrant i have both a low standard and symmetry - corrected rmsd ( < 2.0 ) , thus would be considered successfully docked poses by either metric . among the results , poses in quadrant ii have high standard rmsd values ( 2.0 ) , which would ordinarily be considered a docking failure , but they have low symmetry - corrected rmsd values ( < 2.0 ) . thus , they are false - negatives , or poses that are rescued from failure by the symmetry - correction algorithm . poses in quadrant iii have a high rmsd by both metrics ( 2.0 ) , and are considered to be the true negatives . finally , quadrant iv ( false positives ) can not contain any poses and should always remain empty . populations are binned in 0.2 increments , and are colored from green ( low population ) to red ( high population ) as shown on the right - hand side bar . dashed lines at x = 2 and y = 2 delineate four quadrants : i = true positives , ii = false negatives , iii = true negatives , iv = false positives . from figure 5 , it is evident that no poses are assigned a symmetry - corrected rmsd greater than the standard rmsd , consistent with expectations . the vast majority of poses fall near to the diagonal , as indicated by the thick red band across the heat map . however , while the average difference in rmsd between the two metrics ( rmsdstd rmsdcor ) is only 0.96 , 11 914 ( 13.4% ) poses differ by more than 2.0 , and 5490 ( 6.2% ) poses differ by more than 3.0 . the poses that differ by greater than 3.0 represent the most interesting cases of highly symmetric molecules that the hungarian algorithm was designed to uncover , as discussed in the next section . the vast majority of poses in figure 5 , encompassed within the breadth of the red band , are likely indicative of commonly occurring , smaller corrections to symmetric functional groups . for example , as a point of comparison , in a hypothetical molecule of 25 heavy atoms , a 180 rotation of a single phenyl group would result in relatively small rmsd of 0.96 , while the 180 rotation of two phenyl groups would result in a larger rmsd of 1.36 . to facilitate discussions of differences between standard and symmetry - corrected rmsd , it is convenient to analyze docking outcomes grouped by the four quadrants as shown in table 1 . the results here are further subdivided to specifically look at outcomes wherein only the top - scoring pose was retained for each system ( n = 1043 ) , or all poses that were generated were retained ( n = 88 853 ) . it is important to note that the poses analyzed here follow a clustering protocol such that only a diverse set of poses from each ligand docked to its target will be retained . these poses are diverse in terms of both conformation and orientation within the binding pocket . therefore , given the constraints of diversity , typically only one pose would be expected to be the correct pose , or within 2.0 from the crystallographic reference . in addition , it is expected that a small subset of poses , those that overlay within 2.0 from the reference but inverted over an axis of symmetry , will be rescued by the symmetry - correction algorithm . all remaining poses should be perceived as incorrect , or not within 2.0 from the reference . from the perspective of the dock6 scoring function , the correct poses and the rescued poses are likely to have indistinguishable energy scores , and it is a statistical coin - flip that ultimately determines which one is the top - ranked pose . among the data presented in table 1 , these expectations appear to hold true . specifically , approximately one pose per system , or 1272 ( 1.4% ) , is identified as a true positive ( quadrant i ) . the slight discrepancy between true positive poses ( n = 1272 ) and number of systems ( n = 1043 ) can be attributed to the dock6 sampling algorithm , which may generate two poses that are each less than 2.0 from the crystallographic reference ligand , but greater than 2.0 from each other , in which case both poses would be kept . in addition , 853 poses ( 1.0% ) are identified as false negatives and rescued by the symmetry - correction algorithm ( quadrant ii ) , which is consistent with the hypothesis that only a small subset of poses can overlay with the crystallographic reference , but be inverted over some axis of symmetry . finally , the vast majority , or 86 728 ( 97.6% ) , of all poses are true negatives ( quadrant iii ) . this demonstrates that the ensembles of candidate poses generated by dock6 fully explore the boundaries of the binding pockets and vary largely in conformation and orientation from the crystallographic ligands . as expected , no poses are considered false positives given that symmetry - corrected rmsd can never yield a higher value than standard rmsd ( quadrant iv ) . the need to account for symmetry becomes more important when only the top - scored pose for each system ( n = 1043 ) is examined , as is typical for most applications of docking and virtual screening . while the top pose in table 1 is ranked successfully by both rmsd metrics 67.7% of the time , which is consistent with our laboratory s earlier statistics , the number of poses rescued through symmetry correction increases from 1.0% to 5.0% . therefore , the true pose - reproduction success rate using the flexible ligand docking protocols presented here becomes 72.7% ( 67.7% + 5.0% ) across all 1043 systems in the sb2012 test set . a scatter plot comparing standard and symmetry - corrected rmsd values for the top pose from each system is shown in figure 6 . again , most of the top - scored poses fall near to the diagonal . focusing on quadrants i and ii ( figure 6 , insert ) , specific examples of top - scored poses begin to emerge as individual red points . the 20 most interesting cases ( out of 52 in quadrant ii ) with large standard rmsd and small symmetry - corrected rmsd values are shown in figure 7 . scatter plot of standard rmsd ( x - axis ) vs symmetry - corrected rmsd ( y - axis ) . each red point represents the top - scored pose from systems in the sb2012 test set . the dashed lines plotted by x = 2 and y = 2 delineate the four quadrants . the solid line indicates the diagonal . the bottom insert is a magnification of quadrants i and ii . examples of 20 molecules from a total of 52 rescues among the top - scored poses . the pdb code of the system is shown , followed by the standard rmsd , and in parentheses the symmetry - corrected rmsd . each panel of figure 7 contains the crystallographic reference pose ( in red ) overlaid with the top - scoring docking outcome ( in green ) . some of the poses , especially in the top three rows of figure 7 , are easily identifiable as highly symmetric molecules . the symmetry - correction algorithm has in some cases improved the reported rmsd by as much as 10 , a very significant change . also in figure 7 , there are examples of molecules ( e.g. , 1sg0 and 1sri ) that were not inverted over a major axis of symmetry , but instead the rmsd was improved above the success threshold when accounting for a ring flip ( as described previously ) . importantly , a breakdown by protein families from the test set reveals that although the overall increase in success rate is 5.0% , more dramatic success rate improvements ranging up to 16.7% for specific families are observed ( table 2 ) . particularly encouraging is the 13.3% success rate improvement across the large hiv protease test set ( n = 60 ) containing highly symmetric cyclic - urea based ligands , which provides a challenging test case for docking . of the nine protein families in table 2 for which symmetry - correction appears to offer no benefit ( 0% increase in success ) , five families already yield a standard rmsd success rate of greater than 50% , including the highly accurate results for hiv reverse transcriptase at 95.2% and neuraminidase at 93.0% . in these latter two cases , symmetry - correction the remaining four families in table 2 with standard rmsd success rates of less than 50% are for systems for which docking has been particularly challenging ( see figure 7 in mukherjee et al . ) and symmetry correction here provides no improvement . as described in computational details , we have adapted the hungarian algorithm to enable pruning of molecules during de novo growth to avoid combinatorial explosion . the ultimate goal is a final ensemble of molecules with high diversity and constructed within a reasonable amount of time , that can then be used to guide organic synthesis or to identify related purchasable compounds for experimental testing . in terms of evaluation and validation of our de novo routines , we hypothesize that hungarian - based pruning will increase efficiency in experiments designed to ( 1 ) reconstruct known parent molecules from restricted fragment libraries in their respective binding sites ( i.e. , in less cpu time and with fewer total molecules sampled ) and ( 2 ) yield increased diversity in final molecule ensembles constructed from generic fragment libraries . as shown in figure 8 , the first set of experiments designed to promote ligand growth was evaluated on the basis of cpu - time , number of molecules processed , and tanimoto coefficient computed with respect to each of 103 parent compounds . using the new hungarian algorithm - based pruning heuristic , a sharp decrease in cpu time is observed ( figure 8a ) from approximately 5.4 2.6 h as the number of unmatched atoms ( # -unmatched ) threshold increases from 0 to 10 . similarly , the total number of molecules processed shows a marked decrease from approximately 15k 5k molecules over the same threshold ( figure 8b ) . both of these indicators demonstrate that the de novo experiments are finishing more quickly and more efficiently . importantly , the average best tanimoto coefficient observed during the experiment decreases only gradually from 0.97 0.89 ( figure 8c ) . thus , the parent molecules or analogs that are very similar to the parent molecules are being rebuilt with high frequency in approximately half the time , as was the goal of the experiment . ( a ) cpu time required to complete the de novo experiment , ( b ) total number of molecules considered during the de novo experiment , and ( c ) best tanimoto coefficient to the parent compound plotted with respect to the user - defined # -unmatched threshold . results are averaged over all systems with 7 rotatable bonds from the sb2012 test set ( n = 103 ) . in the second set of experiments , we performed de novo growth using the larger seeded generic libraries ( see computational details ) with a variable threshold of 05 for # -unmatched ( figure 9 ) . the focus here was to test our hypothesis that increasing the # -unmatched threshold in the heuristic will increase diversity among the final molecule ensemble . following de novo growth of new compounds , the diversity among members in each ensemble was determined by computing all the possible pairwise tanimoto coefficients between all molecules constructed . for example , an ensemble of 50 molecules would have 50 50 pairs , minus 50 self self - comparisons , for a total of 2450 tanimoto values . for a given # -unmatched threshold , tanimoto coefficients from different systems were compiled , then histogrammed into discrete bins as shown in in figure 9 . histograms of tanimoto coefficients for all pairwise comparisons within de novo molecule ensembles . results are binned in 0.05 coefficient increments over all systems with 7 rotatable bonds from the sb2012 test set ( n = 103 ) . figure 9 shows six final populations of molecules that each characterize different distributions of tanimoto coefficients . as the distribution shifts to the right , molecules within the ensembles are more similar to one another ( closer to 1.0 ) and thus have less diversity . as the distribution shifts to the left , molecules within the ensembles are less similar to one another ( closer to 0.0 ) and thus have greater diversity . consistent with our expectations , the peak shifted furthest to the right ( corresponding to the least diversity ) occurs when the # -unmatched term is set to 0 ( figure 9 , black line ) . and , the de novo ensemble shifted furthest to the left ( corresponding to the most diversity ) occurs when the # -unmatched term is set to 5 ( figure 9 , purple line ) . these data indicate that , during de novo growth , the hungarian algorithm is successfully identifying chemically similar molecules that overlap sufficiently in cartesian space , then pruning the population to make room for more diversity . in addition to the applications described above , two extended applications of the hungarian algorithm are envisioned : ( 1 ) as a scoring function to rank - order docked ligands from virtual screening based on similarity to a known reference and ( 2 ) as a tool to analyze ligand behavior during a molecular dynamics ( md ) simulation . to illustrate the first extended application , we examined docked results from an enrichment study previously performed in our laboratory , in which balius et al . docked 475 known active and 15 990 decoy compounds to the target epidermal growth factor receptor ( egfr , pdb 1m17 ) . the adapted version of the hungarian algorithm for comparing dissimilar molecules was used to rerank that docked ensemble by comparing each docked pose to the cognate inhibitor ( i.e. , reference ) 4-anilinoquinazoline ( erlotinib ) in its crystallographic binding pose with egfr . the total number of unmatched atoms ( # -unmatched ) and the rmsd between the matched atoms ( rmsd - matched ) were returned for each ligand . we hypothesized that many of the known actives would have significant spatial and chemical overlap with the erlotinib reference , and thus would be ranked early in the list using the hungarian approach , thereby providing good enrichment . enrichment was evaluated using four unique ranking methods : ( 1 ) first by # -unmatched , and in case of a tie , then by rmsd - matched , ( 2 ) first by rmsd - matched , and in case of a tie , then by # -unmatched , ( 3 ) by a combination of the # -unmatched and rmsd - matched as shown in eq 5 , and ( 4 ) the standard dock grid score.5 for the ranking method described in eq 5 , # -ref atoms is the number of non - hydrogen atoms in the reference ligand , and c1 and c2 are constants of 5 and 1 , respectively , chosen such that the two terms are weighted approximately equally , and that greater negative values represent more favorable scores . figure 10 presents the receiver operating characteristic ( roc ) curves derived from each of the four ranked lists along with structural overlays for top - scoring compounds from two of the four methods . while visualization of the roc curves can provide a qualitative assessment of enrichment , results are also quantified below using area under the curve ( auc ) computed across the entire database ( overall enrichment ) , and the number of actives recovered after screening 1% of the database ( early enrichment ) . ( a ) roc curves from docking 475 actives and 15 990 decoys to egfr , then ranking the results by one of four metrics . ( b , c ) crystallographic pose of reference ligand erlotinib shown as gray sticks and transparent surface , identical in both panels , relative to the top four ligands from the ( b ) combined value , shown as orange sticks , or the top four ligands from the ( c ) dock grid score , shown as orange sticks . quantitatively , the auc data from figure 10a reveal the following trend in terms of overall enrichment : theoretical maximum ( 1.00 ) > combined value ( 0.92 ) > # -unmatched ( 0.89 ) > rmsd - matched ( 0.83 ) > dock grid score ( 0.58 ) > theoretical random ( 0.50 ) . all four methods provide substantially better enrichment relative to random , and the three hungarian - based approaches provide enhanced enrichment ( figure 10 black , red , blue lines ) relative to the standard dock grid score ( green line ) , in accordance with our hypothesis . in terms of early enrichment , often considered to be a more useful metric for real - world applications of virtual screening , the number of actives recovered at 1% of the database screened are theoretical maximum ( 165 ) > combined value ( 158 ) > rmsd - matched ( 131 ) > dock grid score ( 113 ) > # -unmatched ( 85 ) > theoretical random ( 5 ) . again , all four methods provide significantly enhanced enrichment relative to random , and two of the three hungarian methods provide enhanced early enrichment relative to the standard dock grid score . upon visual examination , the four top - ranked compounds from the combined hungarian method show much better cartesian- and chemical - space overlap with the erlotinib reference ( figure 10b ) compared to compounds obtained using the dock grid score , which are larger and have less well - overlaid groups ( figure 10c ) . overall , while the effectiveness of the scoring function should be tested further on additional systems , these preliminary data indicate the method shows promise in enriching for compounds that are similar to a known reference molecule . as an illustration of the second extended application , we explored the use of symmetry - corrected rmsd to characterize ligand dynamics as a function of time . we expected that typical sampling of a ligand during an md simulation of a protein ligand complex would lead to the occurrence of ring and other symmetric flips which would be discernible through comparisons of standard vs symmetry - corrected rmsd . figure 11 plots results derived from two protein ligand trajectories from md simulations performed previously in our laboratory : ( 1 ) t4 lysozyme in complex with benzene ( pdb 181l ) , and ( 2 ) -trypsin in complex with 1-(4-amidinophenyl)-3-(4-chlorophenyl)urea ( pdb 1bju ) . here , md coordinate files saved every 1 ps were least - squares fit to the starting frame ( using protein -carbons as the reference ) , followed by the calculation of standard ( figure 11 , red ) and symmetry - corrected ( figure 11 , black ) rmsds for the ligand . as a practical note , in the present example the ligand coordinates were extracted and then postprocessed using the hungarian algorithm code in dock . in theory , however , there is no barrier to incorporating the code directly into an md simulation or analysis package . symmetry - corrected ( black lines ) vs standard ( red lines ) heavy - atom rmsds for two ligands during md simulation . in the first example ( figure 11 , left ) , the benzene ligand was observed to rotate both clockwise and counterclockwise about an axis normal to the plane of the ring ( as indicated by blue arrows ) . although no ring flipping was observed in this simulation , likely because of steric constraints imposed by the binding site , large variations in standard rmsd were measured as a direct result of the rotational motion ( figure 11 , red ) . importantly , the symmetry - corrected rmsd ( figure 11 , black ) effectively corrects for symmetry in the benzene , thus highlighting variations due to either translation or out - of - plane rotations , which in both instances are minimal . in the second example ( figure 11 , right ) , the ligand remained fairly close to its original binding configuration during the md simulation . however , at approximately 550 ps , the 4-chlorophenyl group flipped 180 about a rotatable bond ( as indicated by blue arrows ) . corresponding to this flip , a spike in the standard rmsd was observed , which was not reflected in the symmetry - corrected rmsd ( figure 11 , red vs black ) . and , at approximately 1250 ps , the same ring flips 180 back to the original orientation , and the two rmsd measures reconverge . it should be noted that although this event was not easily detected visually , it was readily apparent in the plotted graphs . overall , although more exhaustive tests should be done , the two extended applications presented in this manuscript suggest the hungarian algorithm will have use in both virtual screening and ligand sampling analysis . in this work , we describe implementation , validation , and application of the hungarian algorithm within the dock6 program to compute symmetry - corrected rmsd . the method is fast , on the order of less than a millisecond per ligand ( figure 4 ) , and therefore adds only a trivial amount of time to on - the - fly flexible ligand docking in dock6 . more importantly , the negligible amount of time required of the algorithm allows its use in de novo design . in all , 88 853 docked poses from the sb2012 test set of 1043 receptor ligand systems were evaluated using the new method . while the majority of poses showed a < 1.0 improvement in rmsd , indicative of local corrections within symmetric functional groups , a smaller but significant set yielded > 3.0 improvement , indicative of higher symmetry corrections ( figure 5 ) . a closer examination of just the top - scoring pose ( table 1 ) from each system revealed 67.7% were true positives , or considered as successfully docked by either metric , and 5.0% were false negatives , or considered to be rescued by the symmetry - correction algorithm , for a total success rate of 72.7% . visualization of specific examples of rescued poses ( figures 6 and 7 ) reveals that the method is behaving as expected . highly symmetric molecules with nearly perfect overlap to crystallographic references but inverted over the axis of symmetry are identified and rescued , and some molecules with local pockets of symmetry are also rescued . analysis of results grouped by specific protein families reveals more dramatic increases in success rate up to 16.7% , including the challenging hiv protease system , for which an increase in success rate of 13.3% over 60 systems was observed ( table 2 ) . the hungarian method was also adapted to be used as a heuristic for comparing spatial and chemical overlap between two dissimilar molecules to aid in pruning in a de novo design version of dock undergoing development . preliminary tests demonstrate that by using the heuristic , sampling remains relatively unaffected , while time and efficiency of computation are significantly improved ( figure 8) . in addition , diversity among molecules in the final growth ensembles is higher , which for these purposes was desirable ( figure 9 ) . if preferred , it would be straightforward to modify the function in order to decrease chemical diversity in growing de novo populations . first , the adapted version of the algorithm was used as a scoring function to rank - order compounds from a large database and thus identify those related to a known reference molecule ( figure 10 ) . compounds with high overlap using the hungarian metric are both spatially and chemically similar to the reference compound . second , the algorithm was used to compute symmetry - corrected rmsds in the course of an md simulation ( figure 11 ) . while additional testing is desirable to more fully establish the utility of the algorithm for these extended applications , the accurate determination of success and failure in docking calculations is essential for evaluation of new sampling routines , scoring functions , protocols , and other code developments . prior to the current release , the program dock contained no method to efficiently correct for symmetry when computing rmsd for small molecule ligands . the source code for this hungarian algorithm implementation is available to registered users of dock as part of the latest release at http://dock.compbio.ucsf.edu .

false negative docking outcomes for highly symmetric molecules are a barrier to the accurate evaluation of docking programs , scoring functions , and protocols . this work describes an implementation of a symmetry - corrected root - mean - square deviation ( rmsd ) method into the program dock based on the hungarian algorithm for solving the minimum assignment problem , which dynamically assigns atom correspondence in molecules with symmetry . the algorithm adds only a trivial amount of computation time to the rmsd calculations and is shown to increase the reported overall docking success rate by approximately 5% when tested over 1043 receptor ligand systems . for some families of protein systems the results are even more dramatic , with success rate increases up to 16.7% . several additional applications of the method are also presented including as a pairwise similarity metric to compare molecules during de novo design , as a scoring function to rank - order virtual screening results , and for the analysis of trajectories from molecular dynamics simulation . the new method , including source code , is available to registered users of dock6 ( http://dock.compbio.ucsf.edu ) .

Introduction Theoretical Methods Computational Details Results and Discussion Conclusions

PMC4145001

common causes of lateral knee pain include lateral meniscus tear , lateral collateral ligament sprain , patellofemoral dysfunction , osteochondral injury , biceps femoris tendonitis , iliotibial band friction syndrome , and osteoarthritis . very few references include myofascial pain syndrome as a source of knee pain , and the contributions of trigger points to knee dysfunction are generally not appreciated . a correlation between lateral tightness and lateral knee pain has been identified in previous studies . lateral knee pain suffered by triathletes frequently is caused by repetitive stress , from cycling and running to the musculotendinous structures that surround the knee . the trauma to the musculotendinous structures can potentially cause bleeding and/or swelling between the tendons and surrounding tissues . microadhesions that develop from this type of soft tissue trauma can influence tissue mobility , alter neurodynamics , and limit joint range of motion . training errors or changes in training routines that have been implicated in overuse knee injuries sustained by triathletes include excessive mileage , a sudden increase in mileage , an abrupt change in training surfaces , hill work , riding in a gear that is too high with a low cadence , equipment changes , and high - level participation without an adequate training base . bicycle fit , running shoes , and running technique are also potential causes for injury . the following is a report of four female amateur triathletes who were referred to the author s physical therapy clinic with chronic lateral knee pain with persistent symptoms lasting longer than seven months . four female amateur triathletes , ages 2743 , developed pain around the right lateral femoral condyle ( figure 1 ) as a result of a change in their training routine . all four athletes consulted with an orthopedic surgeon , were immobilized or restricted from activity for at least two weeks , had normal mri results , completed at least six weeks of physical therapy that consisted of therapeutic modalities and strengthening exercises , and were unable train for more than seven months . each patient filled out a lower extremity functional scale ( lefs ) , global rating of change scale ( grcs ) ( table 1 ) , and underwent a standardized physical exam . the lefs is a questionnaire containing 20 questions about a person s ability to perform everyday tasks . the tasks are ranked on a scale from zero ( extremely difficult or unable to perform ) to four ( no difficulty performing the activity ) . the maximum score is 80 ; the lower the score , the greater the disability . the reliability of the lefs was found to be excellent .94 and the validity supported by comparison with the sf-36 physical function subscale ( r = .80 ) and the sf-36 physical component score ( r = .64 ) . the main purpose of the grcs is to quantify the degree to which the patient has improved or deteriorated over time . grcs involves a single question that asks the patient to rate their change with respect to their condition . with respect to your knee pain , how would you compare yourself now compared to when you first came in for treatment ? in this study , a 15-point scale , ranging from 7 ( a very great deal worse ) , through 0 ( unchanged ) to + 7 ( a very great deal better ) was utilized . the reliability of the grcs was found to be .90 in a cohort of subjects with low back pain . the grcs has shown good validity when compared to the roland morris disability questionnaire ( r = 50 ) , oswestry low back pain disability questionnaire ( r = .78 ) , and the numeric pain rating scale ( r = .49 ) . the physical exam consisted of bilateral active and passive knee rom , valgus and varus stress tests , lachman s test , posterior drawer test , apley s compression test , patellar grind test , knee extension angle ( kea ) ( to measure hamstring flexibility , and the ober s test to measure iliotibial band flexibility . patients were also observed while squatting and jumping to identify the presence of dynamic valgus . baseline and post intervention scores the kea and the ober s tests were measured with a bubble inclinometer ( table 1 ) . the patient was positioned supine on the exam table with one mobilization belt placed across the anterior superior iliac spines and another across the mid - thigh of the left lower extremity . the patient was asked to bring her right thigh toward her chest and support it with both hands clasped behind the knee . the examiner placed a level along the patient s anterior thigh to ensure that the leg was perpendicular to the table . ( in the study performed by gajdosik , a wooden dowel was used to block the subject s thigh to keep the leg perpendicular to the table ) . a bubble inclinometer was placed on the patient s shin and she was asked to actively straighten her lower leg . the measurement was taken at the end of the range of active knee extension , which is the degree of knee flexion from terminal knee extension . a kea angle of 20 has been defined as a cutoff score indicating hamstring muscle tightness . therefore , a kea angle of greater than 20 indicates hamstring tightness ; three of the four athletes exceeded the threshold for hamstring tightness . the ober s test was performed ( figure 3 ) , as described by reese and bandy . the patient was positioned on the examination table on her left side with the hip and knee of the left lower extremity flexed to 45 and 90 , respectively , in order to stabilize the pelvis . the examiner stood behind the patient and with his left hand stabilized the patient s pelvis . with his right hand the examiner reached under the patient s lower leg and grasped the thigh just above the knee , supporting the lower leg with his forearm . the examiner asked the patient to relax all muscles of the lower extremity , while allowing the uppermost limb to drop toward the table through the available hip adduction range of motion . the end position of hip adduction was defined as the point at which lateral tilting of the pelvis was palpated or when the hip adduction movement stopped , or both . in this position , the examiner maintained the alignment and ensured that no tilting of the pelvis nor internal rotation and flexion of the hip occurred , while a second examiner placed the bubble inclinometer over the lateral epicondyle . if the leg was below horizontal it was recorded as a negative number and if it was above horizontal it was recorded as a positive number . reese and bandy found the ober s test to have excellent reliability with an icc value of .90 . after the physical exam was completed , each athlete ran on a treadmill , 0% grade , at her normal running speed until she experienced knee pain sufficient to make her stop running . she was then asked to rate her pain on a numeric scale ( nps ) of zero to ten ( zero representing no pain and ten representing unbearable pain ) just before she stopped running ( table 1 ) . the athletes each filled out a pain diagram illustrating exactly where they were experiencing the pain . the patients were treated by a licensed physical therapist , certified in manual therapy , with over ten years of clinical experience . they were treated three times a week for three weeks and the sessions lasted approximately 40 minutes . the patient was positioned supine on the treatment table , head supported by a pillow , right hip and knee flexed . the author stood on the right side of the patient ; his right hand stabilized the patient s right leg at the knee joint . the anterior border of the iliotibial band ( itb ) was addressed first . the thumb and index finger of the author s left hand contacted the groove between the itb and the quadriceps distally . the stroke followed the border of the itb proximally until the greater trochanter was reached ( figure 4 ) . this technique was repeated for 5 minutes . stroking the anterior border of the itb . the author stood facing the foot of the table ; his left hand stabilized the patient s right leg at the knee joint . the author s thumb and index finger of his right hand contacted the groove between the itb and the distal hamstring . the patient remained supine while muscle bending of the vastus lateralis was performed ( figure 5 ) . the left - hand palm was placed over the vastus lateralis . firmly grasping the proximal aspect of the tibia with the right hand , the author s left hand sheared the vastus lateralis from lateral to medial over the femur . the patient remained supine with the hip and knee flexed approximately 90 and resting over the author s shoulder . contact with the patient was made with the fist at the distal end of the hamstring . the patient was then asked to lie prone and muscle bending of the biceps femoris and gastrocnemius was performed , as described for the vastus lateralis , for 10 minutes . at the end of the treatment the patient was asked to lie supine on the treatment table . the hip and knee were passively flexed and extended for 5 minutes . the reassessment included filling out a lower extremity functional scale ( lefs ) and the global rating of change scale ( grcs ) , remeasuring hamstring and ilotibial band flexibility , and determining running tolerance on the treadmill . the patients were called on the telephone at eight weeks and asked their global rating of change ( figure 6 ) . four female amateur triathletes , ages 2743 , developed pain around the right lateral femoral condyle ( figure 1 ) as a result of a change in their training routine . all four athletes consulted with an orthopedic surgeon , were immobilized or restricted from activity for at least two weeks , had normal mri results , completed at least six weeks of physical therapy that consisted of therapeutic modalities and strengthening exercises , and were unable train for more than seven months . each patient filled out a lower extremity functional scale ( lefs ) , global rating of change scale ( grcs ) ( table 1 ) , and underwent a standardized physical exam . the lefs is a questionnaire containing 20 questions about a person s ability to perform everyday tasks . the tasks are ranked on a scale from zero ( extremely difficult or unable to perform ) to four ( no difficulty performing the activity ) . the maximum score is 80 ; the lower the score , the greater the disability . the reliability of the lefs was found to be excellent .94 and the validity supported by comparison with the sf-36 physical function subscale ( r = .80 ) and the sf-36 physical component score ( r = .64 ) . the main purpose of the grcs is to quantify the degree to which the patient has improved or deteriorated over time . grcs involves a single question that asks the patient to rate their change with respect to their condition . with respect to your knee pain , how would you compare yourself now compared to when you first came in for treatment ? in this study , a 15-point scale , ranging from 7 ( a very great deal worse ) , through 0 ( unchanged ) to + 7 ( a very great deal better ) was utilized . the reliability of the grcs was found to be .90 in a cohort of subjects with low back pain . the grcs has shown good validity when compared to the roland morris disability questionnaire ( r = 50 ) , oswestry low back pain disability questionnaire ( r = .78 ) , and the numeric pain rating scale ( r = .49 ) . the physical exam consisted of bilateral active and passive knee rom , valgus and varus stress tests , lachman s test , posterior drawer test , apley s compression test , patellar grind test , knee extension angle ( kea ) ( to measure hamstring flexibility , and the ober s test to measure iliotibial band flexibility . patients were also observed while squatting and jumping to identify the presence of dynamic valgus . baseline and post intervention scores the kea and the ober s tests were measured with a bubble inclinometer ( table 1 ) . the patient was positioned supine on the exam table with one mobilization belt placed across the anterior superior iliac spines and another across the mid - thigh of the left lower extremity . the patient was asked to bring her right thigh toward her chest and support it with both hands clasped behind the knee . the examiner placed a level along the patient s anterior thigh to ensure that the leg was perpendicular to the table . ( in the study performed by gajdosik , a wooden dowel was used to block the subject s thigh to keep the leg perpendicular to the table ) . a bubble inclinometer was placed on the patient s shin and she was asked to actively straighten her lower leg . the measurement was taken at the end of the range of active knee extension , which is the degree of knee flexion from terminal knee extension . a kea angle of 20 has been defined as a cutoff score indicating hamstring muscle tightness . therefore , a kea angle of greater than 20 indicates hamstring tightness ; three of the four athletes exceeded the threshold for hamstring tightness . measuring knee extension angle ( kea ) . the ober s test was performed ( figure 3 ) , as described by reese and bandy . the patient was positioned on the examination table on her left side with the hip and knee of the left lower extremity flexed to 45 and 90 , respectively , in order to stabilize the pelvis . the examiner stood behind the patient and with his left hand stabilized the patient s pelvis . with his right hand the examiner reached under the patient s lower leg and grasped the thigh just above the knee , supporting the lower leg with his forearm . the examiner asked the patient to relax all muscles of the lower extremity , while allowing the uppermost limb to drop toward the table through the available hip adduction range of motion . the end position of hip adduction was defined as the point at which lateral tilting of the pelvis was palpated or when the hip adduction movement stopped , or both . in this position , the examiner maintained the alignment and ensured that no tilting of the pelvis nor internal rotation and flexion of the hip occurred , while a second examiner placed the bubble inclinometer over the lateral epicondyle . if the leg was below horizontal it was recorded as a negative number and if it was above horizontal it was recorded as a positive number . reese and bandy found the ober s test to have excellent reliability with an icc value of .90 . after the physical exam was completed , each athlete ran on a treadmill , 0% grade , at her normal running speed until she experienced knee pain sufficient to make her stop running . she was then asked to rate her pain on a numeric scale ( nps ) of zero to ten ( zero representing no pain and ten representing unbearable pain ) just before she stopped running ( table 1 ) . the athletes each filled out a pain diagram illustrating exactly where they were experiencing the pain . the patients were treated by a licensed physical therapist , certified in manual therapy , with over ten years of clinical experience . they were treated three times a week for three weeks and the sessions lasted approximately 40 minutes . the patient was positioned supine on the treatment table , head supported by a pillow , right hip and knee flexed . the author stood on the right side of the patient ; his right hand stabilized the patient s right leg at the knee joint . the anterior border of the iliotibial band ( itb ) was addressed first . the thumb and index finger of the author s left hand contacted the groove between the itb and the quadriceps distally . the stroke followed the border of the itb proximally until the greater trochanter was reached ( figure 4 ) . this technique was repeated for 5 minutes . stroking the anterior border of the itb . the author stood facing the foot of the table ; his left hand stabilized the patient s right leg at the knee joint . the author s thumb and index finger of his right hand contacted the groove between the itb and the distal hamstring . the patient remained supine while muscle bending of the vastus lateralis was performed ( figure 5 ) . the left - hand palm was placed over the vastus lateralis . firmly grasping the proximal aspect of the tibia with the right hand , the author s left hand sheared the vastus lateralis from lateral to medial over the femur . the patient remained supine with the hip and knee flexed approximately 90 and resting over the author s shoulder . contact with the patient was made with the fist at the distal end of the hamstring . the patient was then asked to lie prone and muscle bending of the biceps femoris and gastrocnemius was performed , as described for the vastus lateralis , for 10 minutes . at the end of the treatment the patient was asked to lie supine on the treatment table . the hip and knee were passively flexed and extended for 5 minutes . the reassessment included filling out a lower extremity functional scale ( lefs ) and the global rating of change scale ( grcs ) , remeasuring hamstring and ilotibial band flexibility , and determining running tolerance on the treadmill . the patients were called on the telephone at eight weeks and asked their global rating of change ( figure 6 ) . at four weeks , three of the athletes were able to run on the treadmill , 0% grade , at their running speed , for three miles without having to stop due to lateral knee pain . all three athletes stated that they could continue past three miles ; however , the test was stopped due to time constraints . two athletes rated their pain zero on a 10-point scale , and one athlete rated her pain one on a 10-point scale . one of the athletes experienced lateral knee pain after running 0.3 miles , which she rated a 9/10 , and had to stop . the three athletes who were able to run without pain improved 9 to 19 points on the lower extremity functional scale , 3 to 5 points on the global rating of change scale , and they all demonstrated improvement in hamstring and itb flexibility ( table 1 ) . the athlete who was unable to run on the treadmill improved 3 points on the lower extremity functional scale and 0 points on the global rating of change scale , and demonstrated some improvement in hamstring and itb flexibility ( table 1 ) . at eight weeks , the global rating of change for three athletes was a 7 ( a very great deal better ) and they had returned to training with no complaints of lateral knee pain ( table 2 ) . global rating of change scores at baseline , 4 wks , and 8 wks subject underwent arthroscopic surgery to debride a lateral meniscus tear . lateral knee pain encountered by triathletes is frequently caused by repetitive stress to the musculotendinous structures that surround the knee . the onset is usually precipitated by a change in training routine that exceeds the tissues ability to adapt . our four athletes started running hills , took a longer bike ride than usual , increased running mileage too quickly , and rode a new bike that was not fit properly . when they were seen by a physician , they were either immobilized with a brace and/or restricted from activity . histological studies on the effects of immobilization of connective tissue have demonstrated loss of ground substance and water , formation of collagen interfiber cross links , haphazard lying down of newly synthesized collagen , and microadhesions formed from scar tissue that adheres to adjacent nontraumatized connective tissue . these physiological changes may result in restricted tissue mobility , altered neurodynamics , limited joint rom , and ultimately influence function . management of an acute sports injury should include intermittent range of motion to maintain tissue mobility . if a joint must be immobilized , the brace should allow for some motion to occur , or be intermittently removed for active and passive range - of - motion exercises . when an athlete is restricted from normal activity and told to , he or she should also be instructed to perform daily exercises that take the joint through a full range of motion . the goal of soft tissue mobilization is to rehydrate connective tissue , stimulate the production of ground substance , assist in orienting of collagen fibers , and break microadhesions . the result is improved soft tissue mobility , reduced stress on pain sensitive structures , and better function . success of treatment is dependent on an accurate diagnosis , ruling out other common causes for lateral knee pain such as lateral meniscus tear , lateral collateral ligament sprain , patellofemoral dysfunction , osteochondral injury , biceps femoris tendonitis , iliotibial band friction syndrome or osteoarthritis . the techniques themselves are not difficult ; however , determining the cause of the lateral knee pain can be challenging . in this case series one athlete had an intra - articular dysfunction that did not respond to soft tissue mobilization . she ended up having arthroscopic surgery to debride her lateral meniscus . at seven weeks postsurgery after ruling out common causes for lateral knee pain , soft tissue restriction should be considered a potential source of dysfunction . in some cases , soft tissue restriction is overlooked ; athletes go undiagnosed and are limited from sports participation . further studies on the effectiveness of treating lateral knee pain with soft tissue mobilization should be conducted with a larger number of subjects , blinded evaluators , and a randomized control .

study designprospective case series.backgroundthese case reports present results of the treatment of lateral knee pain in four female amateur triathletes . the athletes were referred to the author s clinic with either a diagnosis of iliotibial band friction syndrome or patellofemoral pain syndrome , all four having symptoms for longer than seven months . changes in training routines were identified as the possible cause of the overuse injuries that eventually developed into chronic conditions.interventiontreatment involved soft tissue mobilization of the musculotendinous structures on the lateral aspect of the knee.resultsat four weeks , three of the athletes improved 9 to 19 points on the lower extremity functional scale , 3 to 5 points on the global rating of change scale , and demonstrated improvement in hamstring and iliotibial band flexibility . at eight weeks the global rating of change for these three athletes was a 7 ( a very great deal better ) and they had returned to triathlon training with no complaints of lateral knee pain . one athlete did not respond to treatment and eventually underwent arthroscopic surgery for debridement of a lateral meniscus tear.conclusionsafter ruling out common causes for lateral knee pain such as lateral meniscus tear , lateral collateral ligament sprain , patellofemoral dysfunction , osteochondral injury , biceps femoris tendonitis , iliotibial band friction syndrome or osteoarthritis , soft tissue restriction should be considered a potential source of dysfunction . in some cases soft tissue restriction is overlooked ; athletes go undiagnosed and are limited from sports participation .

INTRODUCTION METHODS Participants Assessment Intervention Reassessment RESULTS DISCUSSION & CONCLUSION

PMC4179116

a total of 123 patients that had undergone ct ( 57 eyes ) or vat ( 66 eyes ) by a single surgeon ( krs ) at the glaucoma clinic of the asan medical center ( seoul , korea ) were included in this retrospective study . ct was performed from march 2008 to december 2010 and vat was performed from january 2011 to july 2011 . patients who were lost before 1 year after surgery or got ocular surgery other than glaucoma surgery during the study period were excluded . if both eyes met the inclusion criteria , only one eye was randomly chosen to be included in the study . the conjunctiva and tenon 's capsule were carefully dissected and the sub - tenon space was widened using blunt scissors for preparation of the bleb . a limbus - based half - thickness scleral flap ( 2.5 mm circumference 2 mm radius ) was then prepared and a 0.2% mitomycin c soaked sponge was applied for 2 minutes under the sub - tenon space . copious irrigation was performed with balanced salt solution ( bss ) to wash out the mitomycin c. ac paracentesis was performed through the clear cornea on the temporal side and ve ( healon , 1% sodium hyaluronate ) was injected into the ac to maintain the proper space . a punch incision was performed to make a sclerotomy into the ac under the partial scleral flap . the conjunctival flap was closed with a 8 - 0 vicryl interrupted suture followed by running sutures . topical corticosteroid ( 1% rimexolone ) , cycloplegics , and an antibiotic ( 0.5% moxifloxacin ) were prescribed for approximately 1 month postoperatively , depending on the eye condition . after ct , approximately half of the ac space was filled with ve applied via the paracentesis site ( fig . also , a small amount of bss was injected into the ac via the same paracentesis site . while injecting bss into the ac when the ac was filled with ve , movement of ve into the bleb space and subsequent elevation of the bleb this procedure facilitated migration of ve from the ac into the bleb space ; thus , the bleb was well - elevated by the underlying ve ( fig . regular follow - up examinations were performed at 1 day , 1 week , 1 month , 3 months , 6 months , 9 months , and 1 year after surgery . if necessary , additional visits were scheduled . during all follow - up visits , the iop was measured using goldmann applanation tonometry , best - corrected visual acuity was determined , and the use of anti - glaucoma medications and the occurrence of complications were monitored . overall success was defined as attainment of an iop greater than 5 mmhg and less than 22 mmhg with or without the use of anti - glaucoma medication , the avoidance of additional glaucoma surgery , avoidance of revision of the original procedure , and the absence of serious complications . complete success was considered to have been achieved when iop could be controlled without the need for anti - glaucoma medication , whereas control of iop with medication constituted qualified success . the success rate , number of postoperative iop - lowering medications , and frequency of additional procedures such as ac reformation and laser suture lysis were compared between the ct and vat groups . kaplan - meier survival analysis with log - rank testing was performed to compare cumulative complete success rates between two groups . all procedures conformed to the declaration of helsinki and the study was approved by the institutional review board of the asan medical center at the university of ulsan ( seoul , korea ) . a total of 123 patients that had undergone ct ( 57 eyes ) or vat ( 66 eyes ) by a single surgeon ( krs ) at the glaucoma clinic of the asan medical center ( seoul , korea ) were included in this retrospective study . ct was performed from march 2008 to december 2010 and vat was performed from january 2011 to july 2011 . patients who were lost before 1 year after surgery or got ocular surgery other than glaucoma surgery during the study period were excluded . if both eyes met the inclusion criteria , only one eye was randomly chosen to be included in the study . the conjunctiva and tenon 's capsule were carefully dissected and the sub - tenon space was widened using blunt scissors for preparation of the bleb . a limbus - based half - thickness scleral flap ( 2.5 mm circumference 2 mm radius ) was then prepared and a 0.2% mitomycin c soaked sponge was applied for 2 minutes under the sub - tenon space . copious irrigation was performed with balanced salt solution ( bss ) to wash out the mitomycin c. ac paracentesis was performed through the clear cornea on the temporal side and ve ( healon , 1% sodium hyaluronate ) was injected into the ac to maintain the proper space . a punch incision was performed to make a sclerotomy into the ac under the partial scleral flap . the conjunctival flap was closed with a 8 - 0 vicryl interrupted suture followed by running sutures . topical corticosteroid ( 1% rimexolone ) , cycloplegics , and an antibiotic ( 0.5% moxifloxacin ) were prescribed for approximately 1 month postoperatively , depending on the eye condition . after ct , approximately half of the ac space was filled with ve applied via the paracentesis site ( fig . also , a small amount of bss was injected into the ac via the same paracentesis site . while injecting bss into the ac when the ac was filled with ve , movement of ve into the bleb space and subsequent elevation of the bleb this procedure facilitated migration of ve from the ac into the bleb space ; thus , the bleb was well - elevated by the underlying ve ( fig . the conjunctiva and tenon 's capsule were carefully dissected and the sub - tenon space was widened using blunt scissors for preparation of the bleb . a limbus - based half - thickness scleral flap ( 2.5 mm circumference 2 mm radius ) was then prepared and a 0.2% mitomycin c soaked sponge was applied for 2 minutes under the sub - tenon space . copious irrigation was performed with balanced salt solution ( bss ) to wash out the mitomycin c. ac paracentesis was performed through the clear cornea on the temporal side and ve ( healon , 1% sodium hyaluronate ) was injected into the ac to maintain the proper space . a punch incision was performed to make a sclerotomy into the ac under the partial scleral flap . the conjunctival flap was closed with a 8 - 0 vicryl interrupted suture followed by running sutures . topical corticosteroid ( 1% rimexolone ) , cycloplegics , and an antibiotic ( 0.5% moxifloxacin ) were prescribed for approximately 1 month postoperatively , depending on the eye condition . after ct , approximately half of the ac space was filled with ve applied via the paracentesis site ( fig . also , a small amount of bss was injected into the ac via the same paracentesis site . while injecting bss into the ac when the ac was filled with ve , movement of ve into the bleb space and subsequent elevation of the bleb this procedure facilitated migration of ve from the ac into the bleb space ; thus , the bleb was well - elevated by the underlying ve ( fig . regular follow - up examinations were performed at 1 day , 1 week , 1 month , 3 months , 6 months , 9 months , and 1 year after surgery . if necessary , additional visits were scheduled . during all follow - up visits , the iop was measured using goldmann applanation tonometry , best - corrected visual acuity was determined , and the use of anti - glaucoma medications and the occurrence of complications were monitored . overall success was defined as attainment of an iop greater than 5 mmhg and less than 22 mmhg with or without the use of anti - glaucoma medication , the avoidance of additional glaucoma surgery , avoidance of revision of the original procedure , and the absence of serious complications . complete success was considered to have been achieved when iop could be controlled without the need for anti - glaucoma medication , whereas control of iop with medication constituted qualified success . the success rate , number of postoperative iop - lowering medications , and frequency of additional procedures such as ac reformation and laser suture lysis were compared between the ct and vat groups . kaplan - meier survival analysis with log - rank testing was performed to compare cumulative complete success rates between two groups . all procedures conformed to the declaration of helsinki and the study was approved by the institutional review board of the asan medical center at the university of ulsan ( seoul , korea ) . the mean age of the participants was 61.7 13.5 years in the ct group and 55.3 15.4 years in the vat group . the proportion of patients with primary open angle glaucoma was significantly higher in the vat group ( ct , 37.6% ; vat , 75.8% ; p < 0.001 ) . the mean preoperative iop was not different between the two groups ( ct , 26.5 10.7 mmhg ; vat , 24.2 9.6 mmhg ; p = 0.206 ) , but the number of iop - lowering medications was greater in the ct group ( ct , 2.84 0.85 ; vat , 2.38 0.79 ; p = 0.002 ) . the mean postoperative iop was significantly lower in the vat group than in the ct group at postoperative 1 day , 1 week , and 1 month . at postoperative 3 months , 6 months , and 1 year , the mean iop was not significantly different between the two groups . the mean postoperative iop data and number of iop - lowering medications are shown in table 2 . the percentage of reuse of iop - lowering medication and the number of iop - lowering medications were significantly higher in the ct group than in the vat group . among postoperative procedures , ac reformation with ve and laser suture lysis were less frequently performed in the vat group although the former did not reach statistical significance ( p = 0.077 , p < 0.001 ) . the cumulative complete success rate was significantly higher in the vat group than in the ct group ( log rank test , p = 0.042 ) ( fig . ve are routinely used in trabeculectomy as well as in other types of intraocular surgery . during ct , ve are used to prevent the sudden collapse of the ac and to protect the corneal endothelium . since ha is the most widely used ingredient of ve and is a known anti - fibrotic agent , it is appropriate to try to expand the role of ve beyond their current use in ct . at the same time , ve within a bleb might function as a valve that can prevent overfiltration because ve would be absorbed more slowly than aqueous humor . the results of the current study indicated that postoperative iop was significantly lower in the vat group in the early postoperative period . despite lower iops in the vat group , the frequency of ac reformation was lower in the vat group , indicating that the insertion of ve in the bleb was effective in maintaining a lower level of iop deep within the ac . although the absorption rate of ve under the subconjunctival space has not been reported , a rounder and more diffusely elevated bleb configuration was observed during the second to third postoperative weeks in eyes which underwent vat , which may suggest an ongoing presence of ve within the bleb . it is speculated that an ongoing presence of ve within the bleb may prevent both ac collapse caused by overfiltration and adhesion of the subconjunctiva . the vat group demonstrated a higher complete success rate and a less frequent use of anti - glaucoma medication . thus , it is speculated that maintaining a lower iop and avoiding a flat ac during the early postoperative period enhanced the surgical success rate in the vat group . one potential explanation is that while the ve migrates from the ac to the bleb the scleral flap edge might be slightly lifted thereby allowing continuous movement of aqueous humor from the ac to the bleb . thus , iop was well controlled without the need for laser suture lysis in most of the cases . as ve remains within the bleb for an extended period , aqueous humor flow appears to be regulated to a certain extent . we began to perform vat cases in early 2011 and found the technique to be superior in terms of iop control and postoperative complication management , and vat has since become our standard treatment protocol . given that we were aware of the superiority of vat according to our preliminary results , it would have been unethical to randomize patients into two groups whereby one group was known to be receiving an inferior treatment . the lack of randomization is still , however , a limitation of the current study . these results should be interpreted cautiously since both the ct and vat groups differed with respect to the proportion of preoperative diagnoses , the amount of glaucoma medication administered was different between the two groups , and the ct procedure was performed earlier than the vat which may have increased the surgeon 's experience in the vat group . the other limitation of the current study would be the relatively shorter follow - up period . seprafilm , a bioresorbable transparent membrane made of ha , is used in trabeculectomy to prevent postoperative adhesion between the conjunctiva and sclera . the use of this product requires additional materials and time . altering one 's surgical technique as done here , however , requires no additional materials or cost and can be performed at the end of ct with a few minor procedural additions and in a short period of time . therefore , we suggest that the technique of augmenting trabeculectomy with ve is effective for improving the surgical success rate .

purposeto evaluate the clinical outcome of viscoelastics ( ve , sodium hyaluronate)-augmented trabeculectomy ( vat , 66 eyes ) and conventional trabeculectomy ( ct , 57 eyes ) for glaucomatous eyes.methodsin the vat group , half of the anterior chamber space was filled with ve via the paracentesis site at the end of ct and a balanced salt solution was injected into the anterior chamber . this procedure induced migration of ve from the anterior chamber into the bleb space ; thus the bleb was elevated with underlying ve . follow - up examinations were performed until 1 year after surgery . success was defined as the attainment of an intraocular pressure ( iop ) greater than 5 mmhg and less than 22 mmhg . if iop was in the range of success without antiglaucoma medication , it was regarded as a complete success.resultsthe mean postoperative iop was significantly lower in the vat group at postoperative 1 day , 1 week , and 1 month . the complete success rate was significantly higher in the vat group ( 89% ) than in the ct group ( 75% ) , though the qualified success rate was not different between the two groups . the number of iop - lowering medications at postoperative 1 year was significantly higher in the ct group ( 1.30 1.08 vs. 0.73 0.98 , p = 0.003 ) . among postoperative procedures , laser suture lysis was required less frequently in the vat group ( p < 0.001).conclusionsplacing ve within the bleb at the end of surgery may result in better iop control and less need for iop - lowering medication without any additional materials , cost , or time .

Materials and Methods Subjects Surgical procedure 1) Conventional trabeculectomy 2) Viscoelastics-augmented trabeculectomy Analysis Results Discussion

PMC4993842

relining dentures with silicones could be very useful for treating patients with ridge atrophy or resorption , bony undercuts , bruxing tendencies , congenital or acquired oral defects requiring obturation , xerostomia , and dentures opposing natural dentition.12 despite its ability to prevent discomfort , inherent surface roughness of resilient liners can cause bacterial and fungal colonization.3 one of the most serious problems concerning resilient denture liners is the loss of adhesion with denture base . this failure can further increase plaque and calculus formation and bacterial growth , hence responsible for denture related stomatitis and even more serious infection in geriatric patients4 . well organized routine cleaning with soap and brush and further with chemical cleanser may control microbial colonization . however , for elderly people , especially for disabled denture wearers , denture cleaning may be challenging.4 therefore , more effective disinfectants that are non - toxic to the patient and to the environment without any negative side effects on dentures and resilient liners are needed . various types of disinfectants were used in dental practice like glutaraldehyde , formaldehyde , sodium hypochlorite , hydrogen peroxide , chlorhexidine and mixture of different chemicals . despite their strong disinfection capabilities , aldehyde solutions are not the material of choice due to their high toxic effects . chlorhexidine and hydrogen peroxide are shown to have a minor effect , especially on candida species.5 sodium hypochlorite solutions are very effective in various concentrations against microorganism . however , prolonged application may cause bleaching of the denture base and denture liners.6 microwave irradiation can also be used to disinfect dentures . however , microwave disinfection might negatively affect the physical properties of the denture base materials.7 ozone is a naturally occurring compound consisting of three oxygen atoms ( triatomic oxygen or trioxygen ) . it can be found in the form of gas in the stratosphere in concentration of 1 - 10 ppm or it can be produced by ozone generators . ozone , in the gaseous and aqueous phase , has been shown to be a powerful and reliable antimicrobial agent against bacteria , fungi , protozoa , and viruses . these properties make ozone an important therapeutic agent in infectious and inflammatory diseases.8 furthermore , ozone decomposes rapidly to o2 and oh radicals in water solutions.910 paa is generated by the reaction of tetraacetylethylenediamine in the presence of an alkaline hydrogen peroxide solution . it has broad antimicrobial efficacy against bacteria , fungi , protozoa , and viruses and it is not affected by protein residues . paa solution decomposes to acetic acid and oxygen in 24 hours and is drained to sewage after use with little to no effect on the environment.1112 peracetic acid - based disinfectants have been widely used in food industry and water treatment facilities , as well as for decontamination and sterilization of thermosensitive medical and hospital equipment and devices such as endoscopes and uv lenses.1113 the research about paa and ozone disinfection is mainly focused on their antimicrobial efficacy . to date , there are no studies in the literature regarding their effect on adhesion of resilient liners to the acrylic resins . therefore , the aim of this study was to evaluate the effect of paa and ozone disinfection on the tensile bond strength ( tbs ) of heat and self - polymerized silicone - based resilient denture liners to denture base acrylic resins . a dumbbell shaped master model was produced according to astm d-412 standards for tensile test of elastic materials . a silicone mold was produced by embedding a master model into the type a silicone impression material ( panasil , 154081 , kettenbach gmbh & co kg , eschenburg , germany ) . modeling wax was melted and poured into the silicone mold . having been set and reached to room temperature , wax patterns were demolded and inspected for any defects and voids . wax patterns were invested in dental stone ( triastone type 4 , 1302055 , triadent , istanbul , turkey ) in a stainless steel metal flask to construct the molds . following the setting of the dental stone , boil - out procedure was performed . 1 ) the heat - polymerized ( meliodent , 10jan051 , heraeus kulzer , hanau , germany ) dumbbell shape specimens were polymerized according to the manufacturer 's instructions in a manner similar to that used in conventional denture construction . two different resilient denture liners from the same manufacturer were selected : mollosil ( mollosil , 150401 , detax , ettlingen , germany ) auto - polymerized and molloplast b ( molloplast b , 140731 , detax , ettlingen , germany ) heat - polymerized silicone - based resilient liner . from the mid segment of the dumbbell shaped resin specimens , the segments to be grinded off were marked with a digital caliper ( mitutoyo corp , kanogawa , japan ) and removed with a diamond bur . the grinded surfaces were conditioned according to the manufacturer recommendations for proper adhesion between acrylic resin and liner . the resilient liners were prepared according to manufacturer instructions , and the upper portions of the flasks were closed after application of the resilient liner . 2 ) for the molloplast b specimens , the adhesive was applied 2 times over the grinded acrylic surfaces and left to bench dry for 60 minutes . the ready - to - use resilient liner was applied over the acrylic surfaces with a clean spatula . the resilient liner and acrylic resin assembly was covered with thin foil and the flask was closed and placed under the hydraulic press for 4 minutes . the flask was opened and the foil and excess material were removed with a sharp scalpel . the flask was located in cold water and heated slowly up to 100 and boiled for 2 hours . after bench cooling , the specimens were removed from the flasks . for the mollosil specimens , the adhesive was applied over the grinded acrylic surfaces and left to bench dry for 1 minute . equal amount of base material and catalyst were mixed for 30 seconds and applied over the acrylic surfaces with a clean spatula . the excess material was removed with a sharp scalpel and the specimens were removed from the flasks . one hundred and twenty dumbbell shaped heat - polymerized denture base resin were prepared : 60 for mollosil and 60 for molloplast b. they were reattached in mid - segment with a 3 mm long resilient liner . the specimens were divided into 2 control groups ( control1 , control7 ) and 4 test groups of paa ( actosed , 0910026 , acto gmbh , braunschweig , germany ) and ozone disinfection ( paa1 , paa7 , ozone1 and ozone7 ; n = 10 ) ( table 1 ) . tensile tests were performed after immersion in distilled water ( 37 ) for 10 minutes ( control1 ) and seven days ( control7 ) for the control groups . the exposure cycle was 10 minutes for paa and 60 minutes for ozone . following each cycle , test specimens were rinsed under running water and stored in distilled water until the next cycle . immediately after one cycle of paa1 and ozone1 and seven cycles ( just one cycle per day ) of paa7 and ozone7 , test specimens were subjected to tensile test using an universal testing machine ( m500 25kn ; testometric co. , rochdale , uk ) with a crosshead speed of 5 mm / sec until debonding or rupture occurred . 3 ) the tested specimens were observed with a magnifying loupe ( 4 ) ( eyemag pro s ; carl zeiss ag , germany ) and the type of failure and tbs values were recorded . the failure types were classified as : adhesive ( no liner remnant on the acrylic surface ) , cohesive ( both acrylic surfaces covered with liner ) and mixed ( acrylic surfaces partially covered with liner ) . measurements of the tbs were analyzed using anova and tukey 's hsd test . the mean tbs values of the control and test groups are presented in table 2 . paa disinfection had no negative effect on the tbs values of mollosil and molloplast b. no statistically significant differences were detected between the mean tbs values of the control and paa tests groups for both materials . on the other hand , single application of ozone disinfectant ( ozone1 ) did not have any negative effect on tbs values of molloplast b ; however , prolonged exposure to ozone ( ozone7 ) decreased its adhesive strength . even distribution was seen in terms of failure type for molloplast b , except control groups in which cohesive type of failure dominated . there are mainly two types of resilient denture liners used in dental practice : acrylic and silicone based liners . acrylic liners have similar composition to acrylic base resin and can create a chemical bond with denture base . after a certain amount of time , softening agents in acrylic liners wash off and harden the liner . on the other hand , silicone based resilient liners can not create a chemical bond with denture base due to their different chemical composition ; yet their softness remains long.1415 despite its relatively weak bonding strength , silicone resilient liner 's greater long term structural stability over acrylic liners made them the material of choice in the current study . various test methods have been used for testing the adhesion between resilient liners and denture base resins including tensile , peel and shear tests.16 the tensile bond strength test has been widely used by researchers.1718 due to lower tear strengths of silicone based resilient liners , the use of tensile bond strength test method to evaluate their adhesion to acrylic resins is recommended.19 in order to achieve bond between silicone liners and denture for a reasonable clinical life , surface conditioning of the denture base is mandatory . although adequate bond strength can be achieved with surface treatments , oral environment with ph , temperature changes and use of various chemicals for cleaning and disinfecting dentures further diminish bond strength and can cause bond failure eventually.20 therefore , disinfectants with little or no effect to the bonding properties of resilient liners to the acrylic are important for long term clinical use . oxidizing agents are usually very effective disinfectants and act by oxidizing the cell membrane of microorganisms , which results in a loss of structure and leads to cell lysis and death.12 a large number of disinfectants operate in this way , including ozone , peracetic acid , and sodium hypochlorite . paracetic acid and ozone , despite their effective disinfecting capabilities , are safe materials for the patient and environment . they are non - toxic and non - allergenic at low concentrations and decompose to water , oxygen , carbon , and oh , which are biocompatible products present in nature.12 the paa has been considered an effective and safe alternative to glutaraldehyde by international reference institutions , such as the us food and drug administration ( fda ) , centers for disease control and prevention ( cdc ) and the association for professionals in infection control and epidemiology ( apic).11 different concentrations of ozone solutions were tested for eradication of different microorganisms in literatures.8212223 generally , as the concentration of ozone increases , the antimicrobial efficacy increases as well . according to studies of arita et al . and nagayoshi et al . concentration of 4 mg / l ozonated water was effective for killing gram - positive and gram - negative oral microorganisms and oral candida albicans in 60 minutes.821 ozonated water can be generated by various ozone generators . hyper medozon comfort ( herrmann apparateau gmbh , germany ) ozone generator was used in the current study for preparation of ozonated water . the concentration was controlled by device and in this study it was adjusted to 4 mg / l . on the other hand , different concentrations of paracetic acid solutions have been used in dental and medical fields depending on the application . in the current study , the paracetic acid concentration was determined by the manufacturers ' instructions . before testing procedure , it was calculated that bond strength test procedure was extended to 60 minutes for each group . during test procedure of control groups , the specimens were left in distilled water to prevent drying , which extended the duration of the specimens in water from 10 minutes to 60 minutes . it has been shown that storing molloplast b and mollosil resilient liners in water one day to one week has no effect on bond strength to acrylic resins.24 therefore , the authors decided not to have two separate control1 ( 10 minutes for paa and 60 minutes for ozone ) groups for mollosil and molloplast b. in the current study , mollosil had lower bond strength than molloplast b , which is in accordance with the previous studies.25 due to uncontrolled polymerization process and cross - link density , self - polymerized polymers generally have weaker mechanical properties.26 besides the differences in the type of polymerization , the compositional differences might alter the characteristics of the resilient liners . the ratio of the matrix to the filler particles of the silicone - based resilient liners directly affects the physical and mechanical properties of the materials . the increase in filler content improves the strength.26 mollosil showed mostly cohesive failures in test and control groups . the inherent lower mechanical properties of the self - polymerized mollosil may be related to the material 's failure . paa disinfection showed no effect on bond strength of resilient liners being tested in the current study . on the other hand , the ozone disinfection affected the bonding strength of resilient liners differently . however , single exposure of molloplast b to ozone solution had no effect on tbs values while multiple exposures decreased tbs . these findings might be explained by the high oxidizing capabilities of ozone solution , which may deteriorate the bond between the liner and denture base . it is well documented that polymerization process continues after deflasking of heat - polymerized silicones.2728 the tbs values of molloplast b in control7 group were significantly higher than those of control1 . it is possible that the polymerization process of molloplast b might have continued after deflasking and increased its bonding strength . there are few studies investigating the effect of disinfection techniques on the bond strength of resilient liners to denture base resins.202930 garcia et al.29 and pisani et al.20 evaluated the influence of denture cleansers on resilient liner adhesion to denture bases by tensile test method and reported no adverse effects . also , machado et al.30 found no negative effects on the peel bond strength of resilient liner to denture base resins when disinfected by microwave irradiation . there are various tbs values in the literature for proper adhesion between resilient liners and acrylic base . it has been reported by different researchers that this value should be between 0.45 - 0.9 mpa.313233 considering these threshold values , all test and controls groups had satisfactory tbs results . nevertheless , prolonged exposure of mollosil to ozone ( mollosil - ozone7 ; 0.9 mpa ) had high risk of decreased tbs to clinically unacceptable values . their use can be objected due to their cost - effectiveness in terms of everyday use for home - care . using ozone generators in dental laboratories or dental clinics seems to be more suitable for disinfection of dentures instead of home - care for now . however , their price and dimensions will decrease and in time they will be more affordable and suitable for daily use . within the limitations of this in vitro study , it has been shown that paa and ozone can be an alternative to disinfection of dentures with resilient liners . while paa disinfection is safe in terms of bonding strength of silicone to acrylic base , multiple disinfection of mollosil with ozone has high risk of de - bonding . further studies are needed for investigation of the longer - term effect of paa and ozone on tbs values of resilient liners to acrylic resins . prolonged ozone exposure might adversely affect the adhesion of self - curing and heat - curing resilient liners to denture base material .

purposethe aim of this study was to evaluate the effect of paracetic acid ( paa ) and ozone disinfection on the tensile bond strength ( tbs ) of silicone - based resilient liners to acrylic resins.materials and methodsone hundred and twenty dumbbell shaped heat - polymerized acrylic resins were prepared . from the mid segment of the specimens , 3 mm of acrylic were grinded off and separated parts were reattached by resilient liners . the specimens were divided into 2 control ( control1 , control7 ) and 4 test groups of paa and ozone disinfection ( paa1 , paa7 , ozone1 and ozone7 ; n=10 ) . while control groups were immersed in distilled water for 10 min ( control1 ) and 7 days ( control7 ) , test groups were subjected to paa ( 16 g / l ) or ozone rich water ( 4 mg / l ) for 1 cycle ( 10 min for paa and 60 min for ozone ) per day for 7 days prior to tensile tests . measurements of the tbs were analyzed using 3-way anova and tukey 's hsd test.resultsadhesive strength of mollosil decreased significantly by application of ozone disinfection . paa disinfection had no negative effect on the tbs values of mollosil and molloplast b to acrylic resin . single application of ozone disinfection did not have any negative effect on tbs values of molloplast b , but prolonged exposure to ozone decreased its adhesive strength.conclusionthe adhesion of resilient liners to acrylic was not adversely affected by paa disinfection . immersion in ozonated water significantly decreased tbs of mollosil . prolonged exposure to ozone negatively affects adhesion of molloplast b to denture base materials .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC2779943

as part of the sci ( 7 ) , several community - based interventions were implemented in ouargaye district from 2004 to 2005 . the community - based interventions overall goal was to guide the community towards the use of quality delivery care during normal and complicated deliveries . the approach was developed after a review of previous community - based interventions towards maternal and child health in burkina faso , among which were the tostan approach and the maternal and neontal health approach in bazega province and koupla district , respectively ( 11 , 12 ) . 1 ) , defined as a process , a movement of the population leading to behavioural changes towards a specific problem , mostly health related but also applicable to other development sectors such as education or agriculture ( 11 ) . the social mobilisation and thus , community mobilisation when restricted to a community is summarised into four interrelated and complementary pillars : advocacy and awareness , social marketing , behavioural change communication ( bcc ) and capacity strengthening ( fig . logic model of community mobilisation strategy applied to the sci , ouargaye district , burkina faso . awareness aims at engaging local traditional leaders , administrative and religious leaders , local associations with the use of skilled care through problem - solving techniques , negotiation , and persuasion and lobbying . it focuses on danger signs during pregnancy and delivery , birth preparedness ( 13 ) , emergency preparedness and quality delivery care . in a context of low literacy , and where there is a value for community self identification and where communities are responsive to recommendations of religious and traditional leaders , it is critical that any intervention in a community meets the needs of local stakeholders and wins their support . empty vessels and understanding and building upon cultural beliefs of care and means of communication is more likely to be effective . community - based interventions through social marketing aim at reducing financial barriers and out of pocket for the use of skilled care . there is no one - size - fits - all approach and communities are encouraged to use acceptable and effective strategies . much has been written on bcc theories such as learning theories , the transtheoretical model , the health belief model , reasoned action and planned behaviour , and social cognitive theory ( 14 ) among others . it is expected that each community use the most relevant , acceptable and effective one to induce change towards safer delivery . in this example of the sci , this model postulates that a person 's or community 's health - related behaviour depends on his perceived risk of outcome ( whether a woman or a family member perceives women at risk for maternal and perinatal deaths if skilled care is not sought during childbirth ) , his perceived severity of the outcome , the perceived benefits of engaging in preventive action and the perceived barriers to taking that action . the model also incorporates cues to action and self - efficacy ( 15 ) and was used for community workshops . lastly is the capacity strengthening pillar which refers to facilitating community problem - solving skills towards skilled delivery care . this process improves ownership of activities and includes analysis and prioritisation of community problems and sustainability of agreed upon consensual decisions . an important part in the capacity building pillar is also prompting household to raise funds for possible emergencies and to promote revenue generating activities . burkina faso is one of the poorest countries in the world , land - locked , situated in the heart of west africa . population is of mostly farmers with very low literacy rate ( 20% nationally but 58% in remote and rural areas ) . the sci activities in ouargaye district included strengthening health facilities ( functional operating room , drugs and equipment ) , recruitment and posting of anaesthetists to ensure round the clock access to life saving interventions at district hospitals , upgrade of competencies of health workers in obstetric best practices , referrals ( ambulance , wireless phone ) , refurbishment of infrastructures and electricity with solar panels . these supply side activities covered two - thirds of health facilities within the district as another financial and technical partner ( unfpa ) had selected the remaining one - third for similar health facility strengthening . the demand side activities however were built around traditional leaders whose areas of influence are beyond health facility boundaries and ultimately covered the whole district . the main steps for implementing the community - based interventions were a baseline survey , an identification of key influential leaders , mobilisation of stakeholders , bcc and monitoring each of which are briefly described below : a baseline qualitative survey : this used individual interviews , focus group discussions , immersion and observation to identify community health seeking behaviours , birthing and weaning practices , the social organisation and social networks , cultural community mechanisms of solidarity , locally valued communication channels and key influential leaders , level of their influence and coverage areas . it also provides an identification of barriers and facilitators to skilled care , community taboos and values and an understanding of the balance of power within the community . the second step was the selection of key influential leaders to be centrepieces of the process of community - based interventions . indeed , societies , worldwide , are characterised by a political and social organisation to ensure peaceful administration and regulation of the society . in mostly rural communities such as those in ouargaye district , there were , besides local administrative officials , people that communities valued , trusted and respected more than administrative officials because of their role model , their wisdom and their religious power among others . there were traditional leaders whom communities listened to , obeyed and respected and who managed all administrative and judicial matters within their areas of influence . by obeying these traditional leaders people had a sense of social belonging to the community . recognising the influence of these traditional leaders and their potential roles for engaging their communities in behavioural change , the sci community mobilisation strategy selected the most influential ones and developed a participatory approach with them . the third step was engaging stakeholders . after identifying among the traditional leaders those willing to fully back the community - based interventions , the next step was to engage a limited number of them in the process . this was done through structured meetings with prominent traditional leaders who , after an identification of main barriers to utilisation of maternal health services and possible solutions , invited their peers and other stakeholders ( health professionals , religious leaders , administrative officials , associations ) and engaged with them in the process ( box 1 ) . meet with a traditional community leader , the most influential and most supportive to the community mobilisation strategy . elicit from the community the main behaviours both from users and providers that prevent the utilisation of maternal health services ( auto - diagnostic , problem - solving techniques ) . discuss with other community leaders the interventions , reach consensus and set up dates for the interventions . the low level of utilisation of maternal health services may be related to several factors , some obvious such as distance to health facility , cost , time and other less obvious such as cultural barriers or perceived quality of care . in partnership with community leaders , a number of culturally acceptable activities were developed building on existing societal mechanisms : community workshops , recruitment and posting of community relay agents in all villages of the intervention areas ( box 2 ) , public concerts with traditional singers , theatres and financial schemes to reduce out - of - pocket expenses during emergencies ( box 3 ) . eligibility criteria of community relay workers ( crw ) be a resident of the community ( male and female are eligible ) be openly elected by the community ( supporters openly chose from potential candidates ) be able to read and write ( for record keeping and report writing purposes ) be between the ages 2540 ( travel in villages and visits to households required ) be motivated and available have means of transportation ( bicycle ) sensitisation about skilled delivery care advocacy for safe delivery peer counselling , family counselling on danger signs , birth preparedness , emergency preparedness reporting and notification of life events ( deaths of mother and newborns , place of deliveries ) identify the main barriers of skilled delivery care with families design of training materials by the skilled care initiative training of trainers by sci and crw implementation of action plan monitoring of action plan . benefit package of crw wheels , tyres and repair materials for bicycle one payment of 30 us dollars for further needs at recruitment moral satisfaction : privilege and honour of being designated by community , and of being a link person between community and sci opportunistic activities ( participation in national immunisation days , filariosis eradication programme , malaria prevention through promotion of bednets ) courtesy visit ( negotiation ) to the identified traditional leader by sci invitation of participants by the traditional leader prepare the site and logistics for the workshop review of sensitisation tools , terms of references for group discussions , planning module and engagement form with the traditional leader training of moderator in problem - solving techniques final checking with the organisers sensitisation ( workshop day 1 ) introduction by the leader theatre portraying community delivery , birthing , weaning practices identification of themes and comments ( danger signs , advantages of antenatal care , hygiene during pregnancy , birth preparedness , post partum care , negative practices , desired behaviours ) animation : traditional music concert community action plan ( workshop day 2 ) finalisation of an action plan formal engagement ( signed document ) of community to implement action plan towards skilled delivery care scale up the interventions using areas of influence of the traditional leaders monitoring and evaluation monitoring of action plan implementation in each village monitoring of deliveries ( home versus facility deliveries ) by village follow up community workshop traditional leaders meeting ( sharing best practices and experiences ) the fifth step was monitoring of action plans and activities , a structured process that started with a preparatory phase and was followed by sensitisation meetings with communities , elaboration of community action plans , a formal and very symbolic engagement from the leaders to implement the action plans , identification of monitoring indicators and feedback processes ( box 3 ) . as part of the sci ( 7 ) , several community - based interventions were implemented in ouargaye district from 2004 to 2005 . the community - based interventions overall goal was to guide the community towards the use of quality delivery care during normal and complicated deliveries . the approach was developed after a review of previous community - based interventions towards maternal and child health in burkina faso , among which were the tostan approach and the maternal and neontal health approach in bazega province and koupla district , respectively ( 11 , 12 ) . 1 ) , defined as a process , a movement of the population leading to behavioural changes towards a specific problem , mostly health related but also applicable to other development sectors such as education or agriculture ( 11 ) . the social mobilisation and thus , community mobilisation when restricted to a community is summarised into four interrelated and complementary pillars : advocacy and awareness , social marketing , behavioural change communication ( bcc ) and capacity strengthening ( fig . logic model of community mobilisation strategy applied to the sci , ouargaye district , burkina faso . awareness aims at engaging local traditional leaders , administrative and religious leaders , local associations with the use of skilled care through problem - solving techniques , negotiation , and persuasion and lobbying . it focuses on danger signs during pregnancy and delivery , birth preparedness ( 13 ) , emergency preparedness and quality delivery care . in a context of low literacy , and where there is a value for community self identification and where communities are responsive to recommendations of religious and traditional leaders , it is critical that any intervention in a community meets the needs of local stakeholders and wins their support . empty vessels and understanding and building upon cultural beliefs of care and means of communication is more likely to be effective . community - based interventions through social marketing aim at reducing financial barriers and out of pocket for the use of skilled care . there is no one - size - fits - all approach and communities are encouraged to use acceptable and effective strategies . much has been written on bcc theories such as learning theories , the transtheoretical model , the health belief model , reasoned action and planned behaviour , and social cognitive theory ( 14 ) among others . it is expected that each community use the most relevant , acceptable and effective one to induce change towards safer delivery . in this example of the sci , this model postulates that a person 's or community 's health - related behaviour depends on his perceived risk of outcome ( whether a woman or a family member perceives women at risk for maternal and perinatal deaths if skilled care is not sought during childbirth ) , his perceived severity of the outcome , the perceived benefits of engaging in preventive action and the perceived barriers to taking that action . the model also incorporates cues to action and self - efficacy ( 15 ) and was used for community workshops . lastly is the capacity strengthening pillar which refers to facilitating community problem - solving skills towards skilled delivery care . this process improves ownership of activities and includes analysis and prioritisation of community problems and sustainability of agreed upon consensual decisions . an important part in the capacity building pillar is also prompting household to raise funds for possible emergencies and to promote revenue generating activities . burkina faso is one of the poorest countries in the world , land - locked , situated in the heart of west africa . population is of mostly farmers with very low literacy rate ( 20% nationally but 58% in remote and rural areas ) . the sci activities in ouargaye district included strengthening health facilities ( functional operating room , drugs and equipment ) , recruitment and posting of anaesthetists to ensure round the clock access to life saving interventions at district hospitals , upgrade of competencies of health workers in obstetric best practices , referrals ( ambulance , wireless phone ) , refurbishment of infrastructures and electricity with solar panels . these supply side activities covered two - thirds of health facilities within the district as another financial and technical partner ( unfpa ) had selected the remaining one - third for similar health facility strengthening . the demand side activities however were built around traditional leaders whose areas of influence are beyond health facility boundaries and ultimately covered the whole district . the main steps for implementing the community - based interventions were a baseline survey , an identification of key influential leaders , mobilisation of stakeholders , bcc and monitoring each of which are briefly described below : a baseline qualitative survey : this used individual interviews , focus group discussions , immersion and observation to identify community health seeking behaviours , birthing and weaning practices , the social organisation and social networks , cultural community mechanisms of solidarity , locally valued communication channels and key influential leaders , level of their influence and coverage areas . it also provides an identification of barriers and facilitators to skilled care , community taboos and values and an understanding of the balance of power within the community . the second step was the selection of key influential leaders to be centrepieces of the process of community - based interventions . indeed , societies , worldwide , are characterised by a political and social organisation to ensure peaceful administration and regulation of the society . in mostly rural communities such as those in ouargaye district , there were , besides local administrative officials , people that communities valued , trusted and respected more than administrative officials because of their role model , their wisdom and their religious power among others . there were traditional leaders whom communities listened to , obeyed and respected and who managed all administrative and judicial matters within their areas of influence . by obeying these traditional leaders people had a sense of social belonging to the community . recognising the influence of these traditional leaders and their potential roles for engaging their communities in behavioural change , the sci community mobilisation strategy selected the most influential ones and developed a participatory approach with them . after identifying among the traditional leaders those willing to fully back the community - based interventions , the next step was to engage a limited number of them in the process . this was done through structured meetings with prominent traditional leaders who , after an identification of main barriers to utilisation of maternal health services and possible solutions , invited their peers and other stakeholders ( health professionals , religious leaders , administrative officials , associations ) and engaged with them in the process ( box 1 ) . meet with a traditional community leader , the most influential and most supportive to the community mobilisation strategy . elicit from the community the main behaviours both from users and providers that prevent the utilisation of maternal health services ( auto - diagnostic , problem - solving techniques ) . discuss with other community leaders the interventions , reach consensus and set up dates for the interventions . the low level of utilisation of maternal health services may be related to several factors , some obvious such as distance to health facility , cost , time and other less obvious such as cultural barriers or perceived quality of care . in partnership with community leaders , a number of culturally acceptable activities were developed building on existing societal mechanisms : community workshops , recruitment and posting of community relay agents in all villages of the intervention areas ( box 2 ) , public concerts with traditional singers , theatres and financial schemes to reduce out - of - pocket expenses during emergencies ( box 3 ) . eligibility criteria of community relay workers ( crw ) be a resident of the community ( male and female are eligible ) be openly elected by the community ( supporters openly chose from potential candidates ) be able to read and write ( for record keeping and report writing purposes ) be between the ages 2540 ( travel in villages and visits to households required ) be motivated and available have means of transportation ( bicycle ) sensitisation about skilled delivery care advocacy for safe delivery peer counselling , family counselling on danger signs , birth preparedness , emergency preparedness reporting and notification of life events ( deaths of mother and newborns , place of deliveries ) identify the main barriers of skilled delivery care with families design of training materials by the skilled care initiative training of trainers by sci and crw implementation of action plan monitoring of action plan . benefit package of crw wheels , tyres and repair materials for bicycle one payment of 30 us dollars for further needs at recruitment moral satisfaction : privilege and honour of being designated by community , and of being a link person between community and sci opportunistic activities ( participation in national immunisation days , filariosis eradication programme , malaria prevention through promotion of bednets ) courtesy visit ( negotiation ) to the identified traditional leader by sci invitation of participants by the traditional leader prepare the site and logistics for the workshop review of sensitisation tools , terms of references for group discussions , planning module and engagement form with the traditional leader training of moderator in problem - solving techniques final checking with the organisers sensitisation ( workshop day 1 ) introduction by the leader theatre portraying community delivery , birthing , weaning practices identification of themes and comments ( danger signs , advantages of antenatal care , hygiene during pregnancy , birth preparedness , post partum care , negative practices , desired behaviours ) animation : traditional music concert community action plan ( workshop day 2 ) finalisation of an action plan formal engagement ( signed document ) of community to implement action plan towards skilled delivery care scale up the interventions using areas of influence of the traditional leaders monitoring and evaluation monitoring of action plan implementation in each village monitoring of deliveries ( home versus facility deliveries ) by village follow up community workshop traditional leaders meeting ( sharing best practices and experiences ) the fifth step was monitoring of action plans and activities , a structured process that started with a preparatory phase and was followed by sensitisation meetings with communities , elaboration of community action plans , a formal and very symbolic engagement from the leaders to implement the action plans , identification of monitoring indicators and feedback processes ( box 3 ) . a baseline survey was conducted in the intervention and comparison districts to assess health indicators , to describe existing interventions and to document barriers and facilitators to skilled care . a description of the study context was done with regard to health human resources , literacy and fertility rates , antenatal coverage and institutional delivery at baseline , and timing of the different components of skilled care activities in intervention and comparison districts . a geo - referenced census was conducted from february to may 2006 in the intervention district ( ouargaye ) and a comparison district ( diapaga ) . further details on the evaluation study design and comparability of districts are presented elsewhere ( 8) . information were collected from all women aged 1249 with experience of pregnancy in the survey referenced period ( 20012005 ) , on the number of pregnancies , pregnancies lasting more than six months , place of delivery , delivery attendant and foetal / newborn survival during the five years prior to the interview ( 20012005 ) , as well as general census data such as household assets , socio - demographic characteristics of household members ( age , sex , occupation , education , marital status , etc . ) . in this study , maternal death was defined as a death of a woman while pregnant or within 42 days of termination of pregnancy , irrespective of the cause of death . this is a time - of - death definition of maternal death , referred to as pregnancy - related death . . caveats of using these operational definitions and an unprecedented five year recall census ( misclassification , underreporting and recall bias ) have been extensively discussed elsewhere ( 8) . description of components of skilled attendance by year ( 7 ) was done over the reference study period 20012005 in intervention and comparison districts . descriptive statistics were used to compare percentages of deliveries in health institutions , maternal mortality ratio and perinatal death rates between the intervention district ( ouargaye ) and the comparison district ( diapaga ) , and before ( 20022003 ) and after ( 20042005 ) implementation of the demand - driven interventions of the sci . in addition , a multivariate analysis was used to assess differentials in maternal and perinatal mortality risk by maternal age , distance from household to nearest health facility , educational level of mothers , household wealth quintiles and by district . a baseline survey was conducted in the intervention and comparison districts to assess health indicators , to describe existing interventions and to document barriers and facilitators to skilled care . a description of the study context was done with regard to health human resources , literacy and fertility rates , antenatal coverage and institutional delivery at baseline , and timing of the different components of skilled care activities in intervention and comparison districts . a geo - referenced census was conducted from february to may 2006 in the intervention district ( ouargaye ) and a comparison district ( diapaga ) . further details on the evaluation study design and comparability of districts are presented elsewhere ( 8) . information were collected from all women aged 1249 with experience of pregnancy in the survey referenced period ( 20012005 ) , on the number of pregnancies , pregnancies lasting more than six months , place of delivery , delivery attendant and foetal / newborn survival during the five years prior to the interview ( 20012005 ) , as well as general census data such as household assets , socio - demographic characteristics of household members ( age , sex , occupation , education , marital status , etc . ) . in this study , maternal death was defined as a death of a woman while pregnant or within 42 days of termination of pregnancy , irrespective of the cause of death . this is a time - of - death definition of maternal death , referred to as pregnancy - related death . . caveats of using these operational definitions and an unprecedented five year recall census ( misclassification , underreporting and recall bias ) have been extensively discussed elsewhere ( 8) . description of components of skilled attendance by year ( 7 ) was done over the reference study period 20012005 in intervention and comparison districts . descriptive statistics were used to compare percentages of deliveries in health institutions , maternal mortality ratio and perinatal death rates between the intervention district ( ouargaye ) and the comparison district ( diapaga ) , and before ( 20022003 ) and after ( 20042005 ) implementation of the demand - driven interventions of the sci . in addition , a multivariate analysis was used to assess differentials in maternal and perinatal mortality risk by maternal age , distance from household to nearest health facility , educational level of mothers , household wealth quintiles and by district . the intervention ( ouargaye ) and comparison ( diapaga ) districts are remote and rural districts located in centre east region and lacked running water , serviceable roads and an effective transport system . prior to the sci , the mean coverage radius of health centres , ratio of physicians to population and ratio of nurses to population were 11 and 16 km , one for 213,000 inhabitants and one for 134,000 inhabitants , and one for 8,300 inhabitants and one for 6,400 inhabitants , respectively , in ouargaye and diapaga . similarly , the fertility rate , the literacy rate , the antenatal care coverage and institutional delivery were , respectively , 7.5 and 6.0% , 21.7 and 18.6% , 63 and 62% , and 18.0 and 24.4% in ouargaye and diapaga districts ( 8) . as shown in fig . 3 , there are similarities between ouargaye and diapaga districts in terms of strengthening health facilities , access to life saving interventions at district hospitals and capacity strengthening for health workers . the main differences are on demand side activities : there was a community - based cost sharing initiative for emergencies and a radio service that the district management used to broadcast weekly sensitisation message to community on reproductive health in comparison district whilst in the intervention district there was more comprehensive and more intensive community mobilisation ( cost sharing initiative for emergencies , advocacy and awareness , bcc and community capacity strengthening ) . this investment in communities through an understanding of their social structure and health seeking behaviours , through identification and partnership with credible community leaders , and through identification of culturally sensitive and locally acceptable approaches to address transport and referrals was the single significant difference between the two districts . timing of supply and demand - side interventions of skilled attendance at delivery in ouargaye and diapaga , burkina faso , 20012005 . births in the comparison district were much less likely to be institutional than births in intervention district . although institutional delivery increased over time in both districts , and although there was a difference at baseline , it significantly increased over time ( p<0.001 ) in both intervention and comparison districts and there was a remarkable and statistically significant 30% increase in intervention district following the implementation of community mobilisation activities compared to 10% increase in diapaga over the same period ( fig . there was a 31.5% reduction in maternal mortality ratios between the period before and after the community mobilisation in intervention district compared with 15.8% reduction in diapaga . although maternal mortality ratios decreased over time in both districts , and although there was a difference between intervention and comparison districts before the community mobilisation , the maternal mortality ratio decreased to a greater extent in ouargaye compared to diapaga ( table 1 ) . mortality ratios in diapaga and ouargaye districts , burkina faso , 20022005 all numbers of deaths reported have been adjusted to proportion of deaths missing date of death ( n=57 ) . a logistic regression was used to assess the risk for perinatal mortality per district and per sci areas within the intervention district ( table 2 ) . in 2005 , the perinatal mortality rates were 27.5 and 33 per 1,000 live birth in ouargaye and diapaga , respectively ( or = 0.75 , ci 0.700.80 ) . similarly , the perinatal mortality rates were 26 and 31.3 per 1,000 live birth in areas covered by sci supply component versus areas not covered in ouargaye district , respectively ( or = 0.72 , ci 0.680.77 ) . these differences persist even after adjusting for maternal age , distance from household to nearest health centre , household assets ownership and place of delivery . multivariate analysis of perinatal mortality risk in ouargaye and diapaga districts , 20012005 the intervention ( ouargaye ) and comparison ( diapaga ) districts are remote and rural districts located in centre east region and lacked running water , serviceable roads and an effective transport system . prior to the sci , the mean coverage radius of health centres , ratio of physicians to population and ratio of nurses to population were 11 and 16 km , one for 213,000 inhabitants and one for 134,000 inhabitants , and one for 8,300 inhabitants and one for 6,400 inhabitants , respectively , in ouargaye and diapaga . similarly , the fertility rate , the literacy rate , the antenatal care coverage and institutional delivery were , respectively , 7.5 and 6.0% , 21.7 and 18.6% , 63 and 62% , and 18.0 and 24.4% in ouargaye and diapaga districts ( 8) . as shown in fig . 3 , there are similarities between ouargaye and diapaga districts in terms of strengthening health facilities , access to life saving interventions at district hospitals and capacity strengthening for health workers . the main differences are on demand side activities : there was a community - based cost sharing initiative for emergencies and a radio service that the district management used to broadcast weekly sensitisation message to community on reproductive health in comparison district whilst in the intervention district there was more comprehensive and more intensive community mobilisation ( cost sharing initiative for emergencies , advocacy and awareness , bcc and community capacity strengthening ) . this investment in communities through an understanding of their social structure and health seeking behaviours , through identification and partnership with credible community leaders , and through identification of culturally sensitive and locally acceptable approaches to address transport and referrals was the single significant difference between the two districts . timing of supply and demand - side interventions of skilled attendance at delivery in ouargaye and diapaga , burkina faso , 20012005 . births in the comparison district were much less likely to be institutional than births in intervention district . although institutional delivery increased over time in both districts , and although there was a difference at baseline , it significantly increased over time ( p<0.001 ) in both intervention and comparison districts and there was a remarkable and statistically significant 30% increase in intervention district following the implementation of community mobilisation activities compared to 10% increase in diapaga over the same period ( fig . there was a 31.5% reduction in maternal mortality ratios between the period before and after the community mobilisation in intervention district compared with 15.8% reduction in diapaga . although maternal mortality ratios decreased over time in both districts , and although there was a difference between intervention and comparison districts before the community mobilisation , the maternal mortality ratio decreased to a greater extent in ouargaye compared to diapaga ( table 1 ) . mortality ratios in diapaga and ouargaye districts , burkina faso , 20022005 all numbers of deaths reported have been adjusted to proportion of deaths missing date of death ( n=57 ) . a logistic regression was used to assess the risk for perinatal mortality per district and per sci areas within the intervention district ( table 2 ) . in 2005 , the perinatal mortality rates were 27.5 and 33 per 1,000 live birth in ouargaye and diapaga , respectively ( or = 0.75 , ci 0.700.80 ) . similarly , the perinatal mortality rates were 26 and 31.3 per 1,000 live birth in areas covered by sci supply component versus areas not covered in ouargaye district , respectively ( or = 0.72 , ci 0.680.77 ) . these differences persist even after adjusting for maternal age , distance from household to nearest health centre , household assets ownership and place of delivery . an analysis of the contexts in intervention and comparison districts before , during and after the sci points to associations between community - driven interventions , increases in institutional births and reductions in maternal and perinatal deaths in ouargaye district compared to diapaga district . although this temporal relationship is not sufficient for attributing cause , the comparability of context and socio - demographic profile in both districts ( 8) , and the similarities of maternal and child health indicators before the sci , and of skilled attendance at delivery interventions with the exception of a comprehensive demand - driven interventions in ouargaye district have empirically led us to the conclusion that community mobilisation was a major contributing factor to the changes observed . these results have also shed some light on configuration and timing of skilled attendance at delivery components . upgrading health facilities , training health personnel and ensuring availability and quality of life saving interventions are requirements for access to skilled care , but uptake of care and effectiveness could not be achieved without comprehensive demand - driven interventions . not only is there an equal importance of both supply and demand - led interventions , but the results indicate that a comprehensive framework for demand - driven activities is needed as opposed to selective community based activities such as the case in the comparison district . although there is no magic bullet , nor a one - size - fits - all ideal community mobilisation approach , closely working with communities by consulting them at all stages of design , planning and implementation of delivery care is critical for achieving reduction in maternal and perinatal mortality . this will require from maternal and child health policy makers and programme managers a comprehensive policy ( fig . 2 ) in working with communities for surveillance , measurements of maternal and newborn adverse outcomes , as well as policy and strategies . although skilled attendance at delivery is widely regarded as the main strategy to reduce maternal and perinatal deaths , there is less certainty on the optimal configurations of interventions and timing for different health system contexts in order to achieve ultimate health gains . this evaluation demonstrates that reaching reductions in maternal and perinatal mortality requires an adequate coverage of life saving interventions and quality of care , but should be equally met with an investment in community mobilisation . in poorest communities such as burkina faso , working in partnership with communities using a comprehensive approach may accelerate progress towards reductions in maternal and perinatal mortality . these demand - driven interventions require an understanding the social structure of local contexts , the political and cultural logic of power , participation and ownership of the process ( hence communities not empty vessels ) for a successful behavioural change strategy towards skilled delivery .

backgroundreducing maternal and perinatal mortality in sub saharan africa remains challenging and requires effective and context specific interventions.objectivethe aims of this paper were to demonstrate the impact of the community mobilisation of the skilled care initiative ( sci ) in reducing maternal and perinatal mortality and to describe the concept and implementation in order to guide replication and scaling up.designsa quasi experimental design was used to assess the extent to which the sci was associated with increased institutional births , maternal and perinatal mortality reduction in an intervention ( ouargaye ) versus a comparison ( diapaga ) district . a geo - referenced census was conducted to retrospectively assess changes in outcomes and process measures . a detailed description of activities , rationale and timing of implementation were gathered from the sci project officers and summarised . data analyses included descriptive statistics and multivariate analyses.resultsat macro level , the main significant difference between ouargaye and diapaga districts was the scope and intensity of the community - based interventions implemented in ouargaye . there was a temporal association relationship before and after the implementation of the demand - driven interventions and a remarkable 30% increase in institutional births in the intervention district compared to 10% increase in comparison district . there was a significant reduction of perinatal mortality rates ( or = 0.75 , ci 0.700.80 ) in intervention district and a larger decrease in maternal mortality ratios in intervention district , although statistical significance was not reached . a comprehensive framework of community mobilisation strategy is proposed to improve maternal and child health in poorest communities.conclusioncontrolling for the availability and quality of health services , working in partnership and effectively with communities , and not for them hence characterising communities as not being empty vessels can have impacts on outcomes . here , in the district with a community mobilisation programme , there was a marked increase in institutional births and reductions in maternal and perinatal deaths .

Process of community mobilisation strategy Community mobilisation: concepts and rationale From concepts to practice: description of the Skilled Care Initiative (SCI) community-based interventions Methods Context, study design and participants Data analysis Results Context and timing of skilled attendance activities in Ouargaye and Diapaga Effects of the Skilled Care Initiative (SCI) community mobilisation on institutional births Effects of the Skilled Care Initiative (SCI) community mobilisation on pregnancy related mortality Effects of the Skilled Care Initiative (SCI) community mobilisation on perinatal mortality Discussion Conclusion

PMC5168470

spinal cord injury ( sci ) refers to a series of spinal injuries caused directly or indirectly by external factors . depending on the segment affected by the injury , symptoms can range from motor and sensory dysfunction , muscle dystonia , and appearance of pathological reflexes . primary sci refers to the injury caused by the external forces acting directly or indirectly on the spinal cord . secondary sci refers to further damage caused by spinal cord compression , generated by edema , hematoma , compressive fractures , and broken intervertebral disc tissue . spinal cord injury is characterized by high morbidity , high cost , and young patient age and it often leads to severe permanent disability . sci not only affects the quality of patients ' lives , but it also adds a burden to the family and the society . the latest statistics showed that the global incidence of spinal cord injury is about 2361009/million people . in usa , about 250,000 people suffer from varying degrees of sci each year , with an annual rate of up to 2850/million . currently , there is a lack of effective clinical therapy to restore nerve function after sci [ 2 , 3 ] . instead , exercise has become the most important quantifiable means for functional recovery [ 4 , 5 ] , and its mechanism has been studied by both clinical doctors and basic researchers . here , we review recent researches on the mechanisms by which exercise training promotes functional recovery after sci . exercise not only directly strengthens paralyzed muscles and promotes motor function recovery but is also promoting brain remodeling , improves spinal microenvironment , and protects damaged distal motoneuron functions , at multiple levels and through various channels , thereby promoting functional recovery . in vitro studies have demonstrated that exercise can induce changes in the local neural circuitry , suggesting that afferent activity can activate cortical cells and promote nerve function remodeling . research on cortical reorganization after peripheral nerve injury also supports the idea of cerebral cortex plasticity . synaptic contact immaturity is implied by the following observations : the length increase and density decrease of dendritic spines within 7 days in adult rats with the semisection but restoring to normal after 28 days . it was shown that exercise after sci could improve functional prognosis and induce cerebral cortex recombination in the somatic region . for example , rats exercises after sci have shown a higher spontaneous firing rate of cortical neurons and enhanced forelimb sensory and sensorimotor stimulation ( i.e. , the forelimb motion projection area has been expanded to the lower limbs ) . in clinical studies , functional magnetic resonance imaging ( fmri ) in patients with cervical spinal cord injury has shown that functional improvement after exercise is related to the degree of activation of the motor cortex . studies using transcranial magnetic stimulation and electroencephalogram ( eeg ) recordings have further confirmed changes in the cortical sensorimotor area . compared with healthy subjects , the sensorimotor cortex area associated with the muscle tissue above the damaged region is expanded in sci patients . positron emission computed tomography ( pet ) studies have shown that , for sci patients , wrist strength exercises increase the activation of the representative area of the contralateral upper limb motor cortex . in addition , case studies have shown that , after complete c6 spinal cord injury , hand exercise can promote functional improvement and increase the representation of the hand muscles in the cerebral cortex . thus , after sci , the functional remodeling of the cerebral cortex occurs to promote functional recovery . moreover , exercise can affect post - sci remodeling of the brain function , through generation of systemic changes , such as improving blood circulation and neuroendocrine regulation and reducing spasticity . studies have confirmed that in animals with passive exercise training after spinal cord transection injury , the plasticity related neurotrophic factor , adenylate cyclase type 1 ( adcy1 ) , and brain - derived neurotrophic factor ( bndf ) increase in the somatosensory cortex , at levels significantly higher than in animals without training . bndf is important for neuronal growth and differentiation , and adcy1 is important for establishment of long - term synaptic plasticity . it was suggested that exercise training could promote brain function remodeling by inducing bdnf expression . graziano et al . found that , in animals with cycling training after thoracic spinal cord transection , tactile stimulation of the hind paw induced a neural response remapped to the cortical regions of front paws and forelegs under deep anesthesia . such training also improved the neurological cortical reorganization corresponding to the lower limbs , despite the interruption of afferent input from these limbs . active exercise can also increase the complexity dendrites in the dentate gyrus and the density of dendritic spines in rats , although the functional significance of these changes is not really clear . similar studies have demonstrated that treadmill training can promote axonal growth [ 19 , 20 ] and lesion proximal collateral sprouting and increase synaptic establishment . other similar findings in mature rat hippocampus have also shown that long - term treadmill training can increase the number of astrocytes and neural stem cells in the lower granular cell layer of the dentate gyrus . exercise stimulates the proliferation of endogenous neural stem cells and generates neurotrophic factors , such as bndf , which in turn regulate neural plasticity and improve motor function [ 22 , 23 ] . some studies have found that early exercise training after the corticospinal motor system injury can restore the contact of corticospinal tract ( cst ) and the movement projection of primary motor cortex ( m1 ) , thus increasing the number of cholinergic intermediate neurons in the ipsilateral and contralateral spinal cord and reducing the physical control disorder . therefore , passive exercise training of the areas below the spinal cord injury level can promote functional reorganization of the cortex . although brain function remodeling is an important mechanism for functional recovery after sci , excessive function remodeling can result in pathological consequences such as illusion of limb sensation and neuropathic pain . therefore , further studies and a deeper understanding of the mechanisms of cortical remodeling are necessary in order to adopt the best strategy after the interruption of sensory afferent pathways . after sci , the distal neuron pathways undergo a wide range of chemical , electrophysiological , and structural changes , which result in spontaneous neurological remodeling . the effects of exercise training on the structure and function of the spinal cord post - sci include reconstruction of the neuronal structure ; cellular proliferation and differentiation ; activation of the metabolism and expression of neuronal substances and neurotrophic factors ; and regulation of the cellular electrophysiological function . experimental studies demonstrate that , after spinal cord injury , the length and density of the distal motor neuron dendrites and the overall neuron cell size are reduced , suggesting that sci can cause secondary damage to injured distal motor neurons . studies have even shown that , after thoracic sci in rats , exercise training can increase the axonal length of the soleus and tibialis anterior motor neurons , within the lumbar spinal cord . research using synaptophysin immunohistochemistry has shown that treadmill training can significantly increase the formation of the lumbar spinal synapses . similarly , stepping training after transection of new born rat spinal cord can cause significant pathway changes and increase motor neuron synapse activation by stimulating primary afferent fibers or white matter tracts . the expression of synaptophysin and psd-95 in the area surrounding the ventral horn of the spinal motor neurons is significantly higher in rats training on the treadmill than in the untrained group . exercise training can activate the motor neuron n - methyl - d - aspartate ( nmda ) receptor , by increasing the expression of bndf and trkb ( tyrosine kinase gene ) in spinal cord . nmda receptors further regulate neuronal survival , dendritic structure , synaptic plasticity , and neuronal circuits . active training can also increase the expression of neurotrophic factors and nestin - gfp around the ependymal area . this promotes ependymal cell proliferation and differentiation into neural precursor cells ( npc ) and further into oligodendrocytes and astrocytes , resulting in nerve regeneration and improved functional recovery . oligodendrocytes play a key role in leading and efficient signal transmission , and in maintaining and protecting a normal neuronal function . the immature oligodendrocytes markers , transferrin and cyclic nucleotide phosphohydrolase ( cnp ) , increased significantly after seven days of active training . the current consensus is that exercise can induce the neurotrophic factor bndf , insulin - like growth factor i ( igf - i ) , and vascular endothelial growth factor ( vegf ) , to promote spinal oligodendrocyte regeneration . active training can also elevate the levels of glial fibrillary acidic protein ( gfap ) , regulate the astrocytes aggregation , and promote astrocytes maturation and differentiation . in rats with treadmill training , the nucleolar area of motoneurons increases and becomes surrounded by basophilic granules . also , the staining intensity of glucose-6-phosphate dehydrogenase increases , indicating a boost in protein synthesis . after training , motoneurons can transport more axonal protein , through either forward or reverse transport , and thus improve the overall adaptability of the motor units . for example , synaptic protein snap25 links synaptic vesicles and presynaptic membrane and motor neuron axons transport synaptic proteins snap25 with high selectivity after training . other proteins , such as the enzyme malate dehydrogenase and the trophic factor calcitonin gene - related peptide , are present in higher amounts in the motoneurons . post - sci cycling training can also raise the amount of phosphocreatine - s6 ( p - s6 ) expressed by intermediate neurons and cause dendrites branching of motor neurons . exercise training can alter the electrophysiological properties of transacted spinal motoneurons , such as the hyperpolarization of resting membrane potential and voltage threshold , the speed increase of action potential , and the increase of after - hyperpolarization potential amplitude of action potential . studies have shown that depolarization of resting membrane potential ( rmp ) and spike trigger level ( stl ) occurs four weeks after complete transection of the thoracic spinal cord . after training , the rmp can become hyperpolarized , thus altering the inhibition of the rubrospinal tract on stepping and promoting functional recovery . stepping training can enhance muscle spindle afferent signals [ 42 , 43 ] , promote aspartate nmda receptors functioning , and increase the amplitude of the incoming signal of the motoneuron synapsis , causing an after - hyperpolarization in the action potential of motoneurons . in addition , the ventrolateral spinal cord white matter ( vlf ) can also induce changes in the electrophysiological activity of motoneurons . in rats with sci , a change in the after - hyperpolarization potential ( ahpd ) affects the rhythm , intensity , and duration of interneuron activity which affects the stepping function . passive exercise can enhance the magnitude of excitatory postsynaptic potentials ( epsp ) , increase the number of motoneurons that accept incoming signal , steer ahpd towards normal level , and restore normal stepping . ultra - microstructural analysis has shown that , after spinal cord transection , exercise training can increase the magnitude of gastrocnemius motor neuron ( mns ) excitatory postsynaptic potential ( epsp ) but has no significant effect on the inhibitory postsynaptic potential ( ipsp ) . spinal cord transection injury results in impaired stepping ability , causes reduced incoming signal from distal motoneuron synapses , enhances inhibitory effects , and thereby inhibits - and -mns activity . studies suggest that this may be related to the significantly more numerous inhibitory f - type enlarged terminals than in the excitatory s - type enlarged terminals . it could also be due to the structural changes occurring in the parallel c - type and m - type enlarged terminals of -mns cell bodies . exercise training can maintain a normal ratio between the excitatory s - type and the inhibitory f - type enlarged terminals of -mns and -mns , as such maintaining the ratio between excitatory and inhibitory signals to improve stepping function . after sci , paralyzed muscles exhibit decreased fiber diameter , reduced voluntary contraction force , decreased metabolism , delayed conversion of slow - twitch to fast - twitch fibers , and a cross - sectional area comprised mainly of type i fibers . currently , it is believed that skeletal muscle atrophy is characterized by lost [ 48 , 49 ] or apoptotic muscle fiber nuclei , suggesting that reduced myoglobin nuclei number leads to nuclear apoptosis . the lost function characterizing muscle atrophy can be restored by several methods , primarily by inducing igf-1 , pax7 , and other molecules that promote myogenic cells ( satellite cells ) activation , proliferation , and differentiation and participating in muscle fibers ( muscle cells ) repair . studies have shown an increase in the soleus igf-1 protein levels after spinal cord injury . after treadmill training , soleus igf-1 shows additional increase , to activate proliferation and differentiation of satellite cells , and increase in the muscle fiber numbers . this implies that exercise training after sci can enhance satellite cell activity and promote muscle fiber formation . endurance training seems to increase terminal branching of nerves at the neuromuscular junction , although results are ambiguous . studies have shown that in cats with sci the expression the levels of myosin heavy chain ( mhc ) in the soleus can be restored after stepping training . after one week of weight - bearing stepping training after sci , the wet weight of the plantaris , medial / lateral gastrocnemius , soleus , and tibialis anterior muscles is significantly reduced . after three weeks of training , twitching and tonic tension peaks in the soleus muscle decrease significantly and the mhc expression in the extensor digitorum longus iix increases . by 10 weeks , the muscle wet weight , contractile properties , and mhc levels returne to baseline levels , except for lg / mg atrophy . although exercise training can improve neurological and skeletal muscle function by modulating the multilevel structures and function of the cerebral cortex , spinal cord , and skeletal muscle following spinal cord injury ( sci ) , current evidence suggests that exercise training has limited efficacy in improving motor function after sci in rodents or cats [ 5760 ] . such inadequate effects are believed to be attributed to insufficient neurotrophic factor production induced by training . therefore , in addition to exercise , more and more combinatory strategies have been investigated . many studies have shown that combined therapy can significantly promote the recovery after sci and relieve spasticity in rats compared to single treatments alone . current strategies focus on combinatorial effects of hematopoietic stem cells , neurotrophic factors , drugs , and electrical or magnetic stimulation . tashiro et al . showed that neural stem cell transplantation combined with treadmill training significantly improved spinal cord pathway conduction and increased central pattern generator activity , resulting in significantly improved motor function . dental pulp stem cell transplantation not only promoted motor function recovery but also significantly reduced lesion cavity and glial scar formation . the secretion of neurotrophic factors can be stimulated in the injured spinal cord by neural stem cell transplantation or exercise training [ 20 , 63 , 64 ] . direct neurotrophic factor application combined with exercise training can also promote functional recovery following sci . combined glial cell line - derived neurotrophic factor ( gdnf ) with early rehabilitation significantly reduced pathological changes and motor dysfunction in patients with sci . other neurotrophic factors such as brain - derived neurotrophic factor ( bdnf ) and neurotrophin-3 ( nt-3 ) can also significantly increase bbb scores , indicating improved functional recovery . the above studies suggest that trophic support from combinatorial treatment is an effective intervention to improve motor recovery after spinal cord injury . in addition , many studies have shown that electrical stimulation or magnetic stimulation combined with exercise training can influence motor function in sci patients . showed that spinal cord electrical stimulation combined with exercise training can induce sustained enhancement of synaptic transmission , thereby improving lumbar anatomical plasticity to promote motor function recovery . demonstrated that functional electrical stimulation combined with treadmill training could activate intraspinal circuits to improve gait control . after sci , load - bearing training combined with functional electrical stimulation can activate ankle flexion to prevent swing phase drag , also reduced swing phase time and improved limb coordination [ 69 , 70 ] . sensory stimulation of the tongue combined with specific training significantly improved the balance and gait of patients with incomplete sci . there are also reports that repetitive transcranial magnetic stimulation combined with plate training can significantly reduce lower limb stiffness in patients with sci . therefore , the combination of electrical or magnetic stimulation and exercise training could significantly improve motor function and maximize functional recovery , which provides a new perspective for clinical rehabilitation . intraperitoneal injection of fluoxetine combined with exercise training significantly increased bdnf in the hippocampus which promoted nerve regeneration and bbb score [ 73 , 74 ] . the combined use of meta - chlorophenylpiperazine ( mcpp ) and quipazine in sci not only improved bbb scores but also improved weight - bearing walking . the implantation of polypyrrole / iodine ( ppy / i ) can protect nerve tissue and promote functional recovery . . showed that combination therapy of chondroitinase abc , neurotrophic factors , and exercise training not only enhanced active motor function recovery by enhancing neuroanatomical plasticity of the descending tracts ( corticospinal tract and 5-ht pathway ) but also significantly reduced the astrocyte proliferation and inflammation around lesions . transplantation of schwann cells and olfactory ensheathing cells in the spinal cord of cats combined with chondroitinase abc treatment significantly improved motor function . drug therapy combined with exercise training is more favorable in the treatment of sci , suggesting that adjuvant drug treatment may have a better prognosis in sci patients in the clinic . in conclusion , exercise training can induce structural and functional changes in the cerebral cortex , spinal cord , and skeletal muscles , thus improving neural and muscular function following spinal cord injury . exercise appears to promote nerve regeneration with functional restoration , to induce corticospinal pathway connectivity , to maintain the functional status of spinal cord neurons , to activate skeletal muscle satellite cells , and to promote muscle fiber regeneration . exercise training combined with other treatments in sci is the future direction with the most promise . more research is needed to optimize the specific training parameters , such as intensity , duration , frequency , and so forth . also , the effect of exercise on sci secondary complications ( e.g. , chronic pain , bladder and gastrointestinal dysfunction , muscle mass loss , osteoporosis , pressure ulcers , joint and muscle pain , fatigue , sleep problems , depression , and temperature control loss ) is rarely discussed in literature and needs further exploration .

the exercise training is an effective therapy for spinal cord injury which has been applied to clinic . traditionally , the exercise training has been considered to improve spinal cord function only through enhancement , compensation , and replacement of the remaining function of nerve and muscle . recently , accumulating evidences indicated that exercise training can improve the function in different levels from end - effector organ such as skeletal muscle to cerebral cortex through reshaping skeletal muscle structure and muscle fiber type , regulating physiological and metabolic function of motor neurons in the spinal cord and remodeling function of the cerebral cortex . we compiled published data collected in different animal models and clinical studies into a succinct review of the current state of knowledge .

1. Introduction 2. Effect of Exercise Training on Cerebral Cortex 3. Effect of Exercise Training on the Structure and Function of Spinal Cord 4. Effect on Neuronal Structure 5. Effect on Cell Biochemistry/Metabolism 6. Effect on Electrophysiological Properties of Motor Neurons 7. Effect of Exercise Training on the Structure and Function of Skeletal Muscles 8. The Effect of Combinatorial Strategy with Exercise

PMC2447742

the interactions of biological macromolecules are critical to their physiological function and dependent on their molecular interaction fields . the electrostatic potential is a molecular interaction field of particular importance for determining the specificity and kinetics of molecular binding . pipsa ( protein interaction property similarity analysis ) ( 1 ) may be used to classify a large number of proteins according to the similarities or dissimilarities in their 3d molecular interaction fields , such as the electrostatic potential ( 2,3 ) . some of the applications of pipsa have been to ww domains ( 4 ) , electron transfer proteins ( 5 ) , ubiquitin conjugating enzymes ( 6 ) , complement control protein modules ( 7 ) and dihydrofolate reductases ( 8) . an extension of pipsa is qpipsa ( quantitative pipsa ) ( 9 ) . qpipsa enables the kinetic parameters of a set of enzymes sharing the same function to be related to the molecular interaction field , e.g. the electrostatic potential , at a functional region of the protein . such a comparison may enable estimation of unknown kinetic parameters for an enzymatic reaction and thereby assist in the modeling and simulation of biochemical pathways ( 9,10 ) . webpipsa allows the user to perform pipsa to compute and compare the electrostatic potentials of a set of structurally related proteins . other web servers such as pce ( http://bioserv.rpbs.jussieu.fr/pce ) ( 11 ) and pfplus ( http://pfp.technion.ac.il ) ( 12 ) also allow the calculation of protein electrostatic potentials . pfplus was designed to extract and display the largest positive electrostatic patch on a protein surface . these web servers , however , only allow the calculation of electrostatic potentials for single proteins . webpipsa on the other hand permits calculation of the electrostatic potentials of a large number of proteins and performs an all - versus - all pairwise comparison of the electrostatic potentials around the entire protein and , optionally , over a user - predefined region . the electrostatic potentials are then computed on a grid by solution of the linearized finite - difference poisson the similarity or dissimilarity of the electrostatic potential of each pair of proteins in the dataset is quantified for a user - defined region by means of similarity indices and distance measures . the results are displayed as an epogram ( tree - like diagram based on electrostatic differences ) and a colored matrix view . the results can be used for the classification of the proteins according to their interaction properties or for the correlation of the molecular interaction fields with functional properties of the proteins . the input required for using webpipsa is a set of protein coordinate files in pdb format . these can be either ( i ) user - supplied or ( ii ) specified by pdb identifier code in the rcsb . the user - supplied structures can be either experimentally determined or generated by comparative modeling techniques using , for example , modeller ( 15 ) or swissmodel ( 16 ) or structures from the respective databases , such as modbase ( 17 ) . an example dataset consisting of triosephosphate isomerases from various species ( 9 ) is provided on the web server and described in detail below . after the structures have been uploaded , the user can choose whether to use the uhbd ( 13 ) or the apbs ( 14 ) software to calculate the electrostatic potentials . example input files for uhbd and apbs are given in the online documentation for webpipsa . at this point , the user is asked to provide an email address to be notified when the pipsa calculation has been completed . the user receives an email with a link to the results page displaying the heat map and a clustered epogram . the email also provides a link to the results directory on the web server containing intermediate and additional results such as the potential grid files and the complete pipsa output . a description of how to visualize electrostatic potential grids from the results directory is given in the online documentation . triosephosphate isomerases from 12 different species are clustered according to the all pairwise distances between electrostatic potentials using a color code from red ( small distance ) to blue ( large distance ) . proteins with similar electrostatic properties are clustered on the top and left side of the graph . the proteins are assigned to a user - defined number of clusters according to their electrostatic potential similarities . the number of clusters can be specified by selecting from a drop down box at the bottom of the result page . here , triosephosphate isomerases from 12 species form four subclusters in a comparison of the electrostatic potentials around the whole protein surface . the type of distance measure [ hodgkin similarity index ( 18 ) , carbo similarity index or average potential differences ( 9 ) ] displayed in the heat map can also be selected from a dropdown box . the distance matrix may also be viewed with the proteins in their input order , without clustering . figure 1.screenshot of part of the results page for the example provided on the webpipsa web server for the comparison of the electrostatic potentials of triosephosphate isomerases from 12 different species . the colored matrix ( heat map ) is shown with color coding corresponding to the distances calculated from the hodgkin similarity indices for the electrostatic potentials . enzymes from species with highly similar electrostatic potentials , such as human , rabbit and chicken , are clustered together ( second subcluster from top / left ) . screenshot of part of the results page for the example provided on the webpipsa web server for the comparison of the electrostatic potentials of triosephosphate isomerases from 12 different species . the colored matrix ( heat map ) is shown with color coding corresponding to the distances calculated from the hodgkin similarity indices for the electrostatic potentials . enzymes from species with highly similar electrostatic potentials , such as human , rabbit and chicken , are clustered together ( second subcluster from top / left ) . after the upload of the protein structures is complete , the user can choose to superimpose the structures using one of two different methods . sup2pdb , starts with one structure ( the template ) and subsequently does a pairwise sequence alignment ( 19 ) between this template structure and the remaining coordinate files . the structures are then superimposed according to the respective alignments . since the outcome of this algorithm depends on the choice of protein template , in the second option , optimized sup2pdb , every coordinate file is considered as a template for superimposing the others . then the most successful superposition based on the maximum number of matched structures and minimal root mean square deviation ( rmsd ) of the superimposed structures is selected . for a large number of protein structures , however , this second option can be time consuming . the protein structures are a subset of the triosephosphate isomerases in the example analysis provided on the web server . the region for comparison of the electrostatic potential can be selected after upload of the coordinate files ( the substrate binding site in the example given ) . these distances are used to cluster the proteins according to the relations between their electrostatic potentials and the clustering is displayed in a tree - like diagram , an epogram . the protein structures are a subset of the triosephosphate isomerases in the example analysis provided on the web server . the region for comparison of the electrostatic potential can be selected after upload of the coordinate files ( the substrate binding site in the example given ) . these distances are used to cluster the proteins according to the relations between their electrostatic potentials and the clustering is displayed in a tree - like diagram , an epogram . the next step in the workflow is to add polar hydrogen atoms to the protein structures using whatif ( 20 ) . standard protonation states at ph 7 are assumed for all residues except for histidine which can be treated as singly or doubly protonated . for the superimposed set of coordinate files , electrostatic potentials are computed automatically using a set of default parameters assuming an ionic strength of 50 mm and a temperature of 300 k. uhbd or apbs can be used to solve the linearized poisson boltzmann equation ( lpbe ) treating the protein as a low dielectric with partial atomic charges embedded in a homogeneous high dielectric continuum representing the solvent . additionally , the user can specify spherical regions within which the electrostatic potentials in the skin are compared . the cartesian coordinates of the center of the sphere and its radius should be input by the user . this option can therefore only be used when the uploaded input structures have already been superimposed . the pipsa software is used to calculate hodgkin and carbo similarity indices of the protein electrostatic potentials as well as average electrostatic potential differences ( the difference in electrostatic potentials in kcal mol e of two proteins divided by the number of grid points in the comparison region where the two protein skins overlap ) described in ( 9 ) . the similarity indices range from 1 ( anti - correlated potential ) , through 0 ( uncorrelated ) to + 1 ( identical potentials ) . these values are converted into distances given by sqrt(2 2*si ) where si is the respective similarity index . these distances thus range from 0 ( identical ) to 2 ( anti - correlated potentials ) . the clustering analysis and generation of tree - like diagrams ( epograms ) on the results page are performed using the statistical program r ( 21 ) . for visualization purposes , both the heat map and epogram representations allow the fast identification of inter - protein relations and classifications for a large set of proteins . one tab is given for the analysis using the entire protein skin and additional tabs are used for each spherical region specified for analysis by the user . a further tab has a java applet with the astex viewer ( 22 ) to allow the user to visually check the superposition of the protein structures as well as the positioning of the spherical regions used for focused comparisons of the electrostatic potentials . as an example , the dataset studied in reference ( 9 ) is provided on the web server for download . these structures of triosephosphate isomerase ( tpi ) from 12 different species are already superimposed ( five of them are shown in figure 2 ) . the example shows how their electrostatic potentials in the vicinity of the active site can be analysed and related to enzymatic kinetic parameters . for example , they have been shown to affect the rate of ligand binding , the affinity of ligand binding and the stability of the transition state . whether a relation between electrostatic potential differences and kinetic parameters or binding affinities exists in a particular case under study depends on the relative contribution of electrostatic interactions to these quantities and the consistency of the structural and the kinetic or thermodynamic experimental data . after the upload of the protein coordinates using an applet , one can request comparison of the electrostatic potentials in specific regions . for the triosephosphate isomerase example , regions may be selected as follows ( with the cartesian coordinates of the center and the radius of a sphere in given ) : the substrate binding site ( region_km : 1.07 , 4.06 , 21.11 , 15 ) where the electrostatic potential correlates with substrate km values , andthe active site ( region_kcat_km : 8.15 , 3.54 , 34.83 , 15 ) where the catalytic turnover occurs and the electrostatic potential influences enzymatic kcat / km values ( 9 ) . the electrostatic potentials are computed for the entire proteins using uhbd . for restricting the region of comparison in case ( i ) , a spherical region of radius 15 around w168 this residue is part of the flexible loop region of the tpis which closes over the active site when the substrate binds . a comparison of the similarity of the electrostatic potentials ( measured by the hodgkin si ) in this region for the five model proteins is shown on the bottom right corner of figure 2 . the five enzymes form two subclusters : tpis from chicken , human and rabbit form one subcluster , whereas yeast and escherichia coli form a distinct second subcluster . this is in agreement with the different substrate km values for the two subclusters : tpis from chicken , human and rabbit possess lower km values ( 0.47 mm , 0.49 mm and 0.43 mm , respectively ) and yeast and e. coli exhibit significantly higher km values ( 1.22 mm and 1.03 mm , respectively ) . for a thorough discussion , thus , the electrostatic potential differences in the tpi loop region can be used as a fingerprint for classifying tpis from various species . such a pipsa analysis can provide insights that are complementary to and not apparent from a protein sequence - based analysis . the substrate binding site ( region_km : 1.07 , 4.06 , 21.11 , 15 ) where the electrostatic potential correlates with substrate km values , and the active site ( region_kcat_km : 8.15 , 3.54 , 34.83 , 15 ) where the catalytic turnover occurs and the electrostatic potential influences enzymatic kcat / km values ( 9 ) . webpipsa is implemented using java servlets and java server pages ( jsp ) running on tomcat 5 ( http://tomcat.apache.org ) . the workflows with significant computational components are implemented as ant scripts ( http://ant.apache.org ) and are launched from a java messaging server ( jms , http://activemq.apache.org ) . this architecture allows the separation of the tomcat web server from the computationally demanding workflows . the user is informed about the current state of the workflow by messages sent from the ant script via the messaging server . all data are stored on the file system and may be removed after 2 weeks without further notice . currently , webpipsa provides a description and categorization of the electrostatic potential differences between the input protein structures . it does not include all the features of the downloadable pipsa software which can be used to analyze other types of molecular interaction field and to select conical as well as spherical regions . these features are planned to be added in future developments of webpipsa . in addition , it is planned to add tools to webpipsa to enable the user to study the relations between protein molecular interaction fields and enzymatic kinetic parameters , as in qpipsa ( 9 ) . on the other hand , webpipsa is much more user - friendly and , therefore , accessible to non - experts . it also has additional analysis features such as the simultaneous display of colored heat maps and epograms as well as protein structure visualization with the astex viewer .

protein molecular interaction fields are key determinants of protein functionality . pipsa ( protein interaction property similarity analysis ) is a procedure to compare and analyze protein molecular interaction fields , such as the electrostatic potential . pipsa may assist in protein functional assignment , classification of proteins , the comparison of binding properties and the estimation of enzyme kinetic parameters . webpipsa is a web server that enables the use of pipsa to compare and analyze protein electrostatic potentials . while pipsa can be run with downloadable software ( see http://projects.eml.org/mcm/software/pipsa ) , webpipsa extends and simplifies a pipsa run . this allows non - expert users to perform pipsa for their protein datasets . with input protein coordinates , the superposition of protein structures , as well as the computation and analysis of electrostatic potentials , is automated . the results are provided as electrostatic similarity matrices from an all - pairwise comparison of the proteins which can be subjected to clustering and visualized as epograms ( tree - like diagrams showing electrostatic potential differences ) or heat maps . webpipsa is freely available at : http://pipsa.eml.org .

INTRODUCTION webPIPSA USAGE webPIPSA WORKFLOW webPIPSA EXAMPLE TECHNICAL OVERVIEW CONCLUSIONS AND OUTLOOK

PMC4519040

pa63 prepores were prepared following the described procedures . for negative - stain em , 2 l of 0.1% polylysine solution ( polysciences ) was first applied to a glow - discharged grid covered with carbon film and then removed by blotting with filter paper in 2 min . the polylysine treatment produced different orientations of particles on em grids , therefore overcoming the problem of preferred orientation for the pa pore . immediately after removal of polylysine , 2 l of pa prepore ( ~50 g / ml ) was applied to the grid and incubated for 1 min . the grid was then washed with the high ph buffer ( 50 mm hepes , 50 mm nacl , ph 8.0 ) twice followed by two washes with the low ph buffer ( 50 mm naoac , ph 5.0 , 0.05% igepal ca-630 ) to induce the conversion of prepore to pore . after removal of excess buffer , the grid was stained with 0.8% ( w / v ) uranyl formate . for cryoem , quantifoil r1.2/1.3 holey grids were covered with a thin layer of continuous carbon film a day before use . the procedure for pore induction on cryoem grids was carried out following the same procedure for the negative - stain em except that the last step of staining was not used . about 1.5 l of low ph buffer was left on the grids before they were transferred into an fei vitrobot mark iv . the grids were then blotted and flash - frozen in liquid ethane in the vitrobot at 100% humidity . negative - stain em micrographs were acquired with leginon automation software and a tietz f415mp 16-megapixel ccd camera at 68,027 magnification in an fei tecnai f20 electron microscope operated at 200 kv . the micrographs were saved by 2 binning to yield a pixel size of 4.4 . frozen - hydrated cryoem grids were loaded into an fei titan krios electron microscope operated at 300 kv for automated image acquisition with leginon . cryoem micrographs were recorded on a gatan k2 summit direct electron detection camera using the electron counting mode at 22,500 nominal magnification ( calibrated pixel size of 1.28 on the sample level ) and defocus values ranging from 1.8 to 5.1 m . the dose rate on the camera was set to ~8 e / pixel / s . the total exposure time was 8 s and fractionated into 32 frames of subimages with 0.25 s exposure time for each frame . frame images were aligned and averaged for correction of beam - induced drift using the gpu - accelerated program from yifan cheng 's lab . the average images from all frames were used for defocus determination and particle picking , and those from the first 16 frames ( corresponding to ~20 e / total dose on sample ) were used for 2d and 3d image classification . in total , 12,416 micrographs were taken in a continuous session . the best 7,062 micrographs were selected for the following in - depth data processing . for negative - stain em single particle reconstruction , 140,775 particles were picked from 1,115 negative - stain em micrographs using the batchboxer program of eman . the defocus value of each micrograph was determined by ctffind and particles were corrected for contrast transfer function ( ctf ) by phase - flipping with the corresponding defocus and astigmatism values using bsoft . the refinement was then performed with 7-fold symmetry using eman . for cryoem single particle reconstruction , 21,200 particles ( 256256 pixels ) were initially picked by hand from 1,928 micrographs and subjected to auto - refinement by relion using the negative - stain em map obtained above as the initial model . the resulting cryoem map ( ~4 resolution ) was used to generate projections that were then served as templates to pick 259,719 particles from all of the 7,062 micrographs using the batchboxer of eman . in our procedure , the defocus values of the micrographs were determined by ctffind and particles were corrected for ctf by phase - flipping using bsoft . the particles were processed with 2d and 3d classifications using the recommended procedures of relion ( http://www2.mrc-lmb.cam.ac.uk/relion/index.php/recommended_procedures ) . 2d class averages and 3d class reconstructions were inspected and those without high - resolution and interpretable features were considered as " bad " classes . the c7 symmetry was applied throughout the 3d classification and 3d auto - refinement . to maximize usable signals from the frame images acquired with the k2 summit camera , we employed the resolution and dose - dependent model of radiation damage recently introduced in relion-1.3 in the following steps . first , the particle images averaged from all 32 frames with whole - image drift correction were used for a preliminary 3d auto - refinement . second , particle images from individual frames were used to calculate translational alignments for the particle - based drift correction . a running average of seven frames , a standard deviation of one pixel , and fitting of linear tracks through the translations for all running averages were used for the optimal translational alignment following the suggested protocol of relion . last , particle images from frame 3 to frame 27 ( ~30 e / total dose on sample ) were translated using the above optimal alignment and weighted with different b - factors as estimated from the single - frame reconstructions to generate optimal " shiny " average images . application of this procedure to the above selected particles yielded 60,455 " shiny " particles . these " shiny " particles were then subjected to 3d auto - refinement in relion to generate the final cryoem map . relion post - processing with a soft auto - mask estimated a resolution of 2.9 by the gold - standard fsc at 0.143 criterion and a b - factor of 95 , and the post - processing without any mask reported a resolution of 3.3 . the accuracies of rotation and translation reported by relion 3d auto - refinement were 1.67 and 1.01 . for visualization and atomic model building , the cryoem map was sharpened and low - pass filtered by relion post - processing using the above - mentioned b - factor and resolution . local resolution was calculated by resmap using the two cryoem maps independently refined from halves of data . 3d classification and auto - refinement also identified a subset of 21,632 particles that led to a cryoem map at 3.6 resolution which is in the pore state but lacks the density corresponding to the 14-stranded barrel , i.e. only has the corolla and calyx of the pa pore . de novo atomic model building of the pa pore except its domain 4 was carried out on the cryoem map at 2.9 resolution using coot . note this procedure only improved the atomic model and did not modify the cryoem map . briefly , the cryoem map was put into an artificial crystal lattice to calculate its structure factor using the em_map_to_hkl.inp utility program in cns . the amplitudes and phases of the structural factor the restraints of ramachandran , secondary structure , and non - crystallographic symmetry were used in the refinement . the available crystal structures of pa83 monomer ( e.g. pdb i d : 1acc and 3tew ) and pa63 heptameric prepore ( pdb i d : 1tzo ) are highly similar to each other , therefore 3tew , which has the highest resolution among them , was used for structural comparison with our cryoem structure of pa pore in most situations and 1tzo was used when the inter - protomer interaction or domain movement were considered . for negative - stain em , 2 l of 0.1% polylysine solution ( polysciences ) was first applied to a glow - discharged grid covered with carbon film and then removed by blotting with filter paper in 2 min . the polylysine treatment produced different orientations of particles on em grids , therefore overcoming the problem of preferred orientation for the pa pore . immediately after removal of polylysine , 2 l of pa prepore ( ~50 g / ml ) was applied to the grid and incubated for 1 min . the grid was then washed with the high ph buffer ( 50 mm hepes , 50 mm nacl , ph 8.0 ) twice followed by two washes with the low ph buffer ( 50 mm naoac , ph 5.0 , 0.05% igepal ca-630 ) to induce the conversion of prepore to pore . after removal of excess buffer , the grid was stained with 0.8% ( w / v ) uranyl formate . for cryoem , quantifoil r1.2/1.3 holey grids were covered with a thin layer of continuous carbon film a day before use . the procedure for pore induction on cryoem grids was carried out following the same procedure for the negative - stain em except that the last step of staining was not used . about 1.5 l of low ph buffer was left on the grids before they were transferred into an fei vitrobot mark iv . the grids were then blotted and flash - frozen in liquid ethane in the vitrobot at 100% humidity . negative - stain em micrographs were acquired with leginon automation software and a tietz f415mp 16-megapixel ccd camera at 68,027 magnification in an fei tecnai f20 electron microscope operated at 200 kv . the micrographs were saved by 2 binning to yield a pixel size of 4.4 . frozen - hydrated cryoem grids were loaded into an fei titan krios electron microscope operated at 300 kv for automated image acquisition with leginon . cryoem micrographs were recorded on a gatan k2 summit direct electron detection camera using the electron counting mode at 22,500 nominal magnification ( calibrated pixel size of 1.28 on the sample level ) and defocus values ranging from 1.8 to 5.1 m . the dose rate on the camera was set to ~8 e / pixel / s . the total exposure time was 8 s and fractionated into 32 frames of subimages with 0.25 s exposure time for each frame . frame images were aligned and averaged for correction of beam - induced drift using the gpu - accelerated program from yifan cheng 's lab . the average images from all frames were used for defocus determination and particle picking , and those from the first 16 frames ( corresponding to ~20 e / total dose on sample ) were used for 2d and 3d image classification . in total , 12,416 micrographs were taken in a continuous session . for negative - stain em single particle reconstruction , 140,775 particles were picked from 1,115 negative - stain em micrographs using the batchboxer program of eman . the defocus value of each micrograph was determined by ctffind and particles were corrected for contrast transfer function ( ctf ) by phase - flipping with the corresponding defocus and astigmatism values using bsoft . the refinement was then performed with 7-fold symmetry using eman . for cryoem single particle reconstruction , 21,200 particles ( 256256 pixels ) were initially picked by hand from 1,928 micrographs and subjected to auto - refinement by relion using the negative - stain em map obtained above as the initial model . the resulting cryoem map ( ~4 resolution ) was used to generate projections that were then served as templates to pick 259,719 particles from all of the 7,062 micrographs using the batchboxer of eman . in our procedure , the defocus values of the micrographs were determined by ctffind and particles were corrected for ctf by phase - flipping using bsoft . the particles were processed with 2d and 3d classifications using the recommended procedures of relion ( http://www2.mrc-lmb.cam.ac.uk/relion/index.php/recommended_procedures ) . 2d class averages and 3d class reconstructions were inspected and those without high - resolution and interpretable features were considered as " bad " classes . the c7 symmetry was applied throughout the 3d classification and 3d auto - refinement . to maximize usable signals from the frame images acquired with the k2 summit camera , we employed the resolution and dose - dependent model of radiation damage recently introduced in relion-1.3 in the following steps . first , the particle images averaged from all 32 frames with whole - image drift correction were used for a preliminary 3d auto - refinement . second , particle images from individual frames were used to calculate translational alignments for the particle - based drift correction . a running average of seven frames , a standard deviation of one pixel , and fitting of linear tracks through the translations for all running averages were used for the optimal translational alignment following the suggested protocol of relion . last , particle images from frame 3 to frame 27 ( ~30 e / total dose on sample ) were translated using the above optimal alignment and weighted with different b - factors as estimated from the single - frame reconstructions to generate optimal " shiny " average images . application of this procedure to the above selected particles yielded 60,455 " shiny " particles . these " shiny " particles were then subjected to 3d auto - refinement in relion to generate the final cryoem map . relion post - processing with a soft auto - mask estimated a resolution of 2.9 by the gold - standard fsc at 0.143 criterion and a b - factor of 95 , and the post - processing without any mask reported a resolution of 3.3 . the accuracies of rotation and translation reported by relion 3d auto - refinement were 1.67 and 1.01 . for visualization and atomic model building , the cryoem map was sharpened and low - pass filtered by relion post - processing using the above - mentioned b - factor and resolution . local resolution was calculated by resmap using the two cryoem maps independently refined from halves of data . 3d classification and auto - refinement also identified a subset of 21,632 particles that led to a cryoem map at 3.6 resolution which is in the pore state but lacks the density corresponding to the 14-stranded barrel , i.e. only has the corolla and calyx of the pa pore . de novo atomic model building of the pa pore except its domain 4 was carried out on the cryoem map at 2.9 resolution using coot . note this procedure only improved the atomic model and did not modify the cryoem map . briefly , the cryoem map was put into an artificial crystal lattice to calculate its structure factor using the em_map_to_hkl.inp utility program in cns . the amplitudes and phases of the structural factor the restraints of ramachandran , secondary structure , and non - crystallographic symmetry were used in the refinement . the available crystal structures of pa83 monomer ( e.g. pdb i d : 1acc and 3tew ) and pa63 heptameric prepore ( pdb i d : 1tzo ) are highly similar to each other , therefore 3tew , which has the highest resolution among them , was used for structural comparison with our cryoem structure of pa pore in most situations and 1tzo was used when the inter - protomer interaction or domain movement were considered . some representative top - view and side - view particles are selected with circles and squares , respectively . b , a full - size drift - corrected cryoem micrograph of 3710 3710 pixels of pa pore particles acquired from a gatan k2 summit direct electron detection camera , and at 300 kv accelerating voltage , 2.7 m defocus , and a total dose of 39 e / . h , superimposition of representative regions of the cryoem map ( mesh ) with the atomic model ( stick ) , including helix ( e ) , strand ( f ) , loop ( g ) , and ca ions ( green spheres in h ) . a , gold - standard fourier shell correlation ( fsc ) between two independently refined maps with an auto - mask that was corrected by phase randomization . b , fsc of the final atomic model versus the final cryoem map ( black ) ; of a model refined in the first of the two independent maps used for the gold - standard fsc versus the same map ( red ) and versus the second independent map ( blue ) . c , surface view and cut - through view of the unsharpened cryoem map colored by local resolution estimated by resmap . a , the crystal structure of domain 4 ( purple ribbons ) from pa prepore ( pdb i d : 1tzo ) fits the cryoem map ( grey surface ) of pa pore with a good agreement ( cross - correlation coefficient : 0.91 ) . domain 4 is shifted inwards as a rigid - body for 4 from the prepore conformation ( dark cyan ) to the pore conformation ( purple ) . the host cell receptor ( tem8 or cmg2 ) of pa that binds to domain 4 ( grey ribbons ) is schematically illustrated . a , comparison of the -clamp in the pa pore with its corresponding region in the pa prepore . the residues asp426 and phe427 missing in the crystal structure of the pa63 prepore ( pdb i d : 1tzo ) are modeled based the crystal structure of the pa83 monomer ( pdb i d : 1acc ) . b , close - up top view of the -clamp region in the pa pore . the hydrogen bonds between ser428 , asn399 , and lys397 , which form a chain tethering the 2728 and 210211 loops together , are depicted with dashed lines . a , superimposition of domains 2c of the pa pore and the crystal structure of the pa heptameric prepore ( pdb i d : 1tzo ) . the dashed boxes highlight the conformational difference of the 210211 loop between these two structures . residues asp426 and phe427 are not solved in the crystal structure of pa prepore due to their flexibility . b , superimposition of domains 2c of the pa pore and the pa octameric prepore ( pdb i d : 3hvd ) . note the similarity of the 210211 loop ( dashed box ) between these two structures . c , superimposition of domains 2c of the pa pore and the pa prepore ( monomer ; pdb i d : 3tew ) . d f , close - up views of the rearrangements of the 210211 loop ( d ) , the 2728 loop ( e ) , and the 2526 and 212213 loops ( f ) during the conversion from prepore to pore . val377 and leu378 of the 2526 loop and val455 and tyr456 of the 212213 loop are flipped upside down to obtain the strand conformation in the pa pore , leading to an extension of 26 and 212 ( f ) . cryoem densities ( mesh ) corresponding to these loops are displayed to the right of d f with atomic models superimposed , showing unambiguous atomic modeling . the superimpositions of the cryoem map and the atomic model are shown in views different from the left panels for clarity . the conformational changes of these loops result in a more compact domain 2c with a decrease of 567 in its surface area . a , surface views of two neighboring protomers ( the same domains in the two protomors are in different shades of the same colors ) of the pa pore and the pa prepore ( pdb i d : 1tzo ) visualized from inside pa heptamer . domains 1 ' , 2c , 2s , and 3 are colored differently and domain 4 is not shown . the inter - protomer interface in the pa prepore is largely formed by domains 1 ' and 3 and domain 2 only contributes to this interface by its membrane insertion loop and c - terminal region ( 213 , 214 , and 23 ) . in the pa pore , the convergence of domain 2c creates an inter - protomer interface without any gap , with an increase of interface area on domain 2c from 1,247 of the pa prepore to 2,106 of the pa pore as calculated using the pisa ( http://www.ebi.ac.uk/pdbe/pisa/ ) . additionally , formation of the barrel also leads to extensive contacts , creating a new interface area of 1,195 between two protomers . b , schematic of the conversion of 22 , 23 , 21 , and the membrane insertion loop of the pa prepore to 22s and 23s of the pa pore . hydrogen bonds between 22 and 23 , which are depicted with dashed lines , are maintained during the conversion . by contrast , 21 and the connecting loops have to be fully unfolded and converted into strands that collectively assemble the barrel of the pa pore . although the detailed events of barrel formation are not yet clear , it is likely that assembly starts from the top in a zipper - like manner . a favorable scenario is that the convergence of domains 2c would place the top ends of the strands close to each other to form a short barrel , which could extend by pulling more residues together via formation of ordered hydrogen bonds until it reaches the bottom end . it is less favorable that the assembly starts from other regions because disordered hydrogen bonds and hydrophobic interactions could generate enormous non - productive , possibly irreversible pairings between strands . b , cross - section side view of superimposition of the unsharpened cryoem map with the atomic model of the intact pa pore , showing the cryoem map has the same conformation as the pa pore except for the absence of the 14-stranded barrel . c , gold - standard fsc ( with an auto - mask that was corrected by phase randomization ) between two independently refined maps . d , top view of the -clamp region of the cryoem map ( mesh ) lacking the barrel superimposed with the atomic model of the pa pore ( ribbons ) , showing correct assembling of the -clamp in the cryoem map .

summaryanthrax toxin , comprising protective antigen ( pa ) , lethal factor ( lf ) and edema factor ( ef ) , is the major virulence factor of bacillus anthracis , an agent that causes high mortality in human and animals . pa forms oligomeric prepores that undergo conversion to membrane - spanning pores by endosomal acidification , and these pores translocate the enzymes lf and ef into the cytosol of target cells1 . pa is not only a vaccine component and therapeutic target for anthrax infections but also an excellent model system for understanding the mechanism of protein translocation . based on biochemical and electrophysiological results , researchers have proposed that a -clamp composed of phe427 residues of pa catalyzes protein translocation via a charge - state dependent brownian ratchet29 . although atomic structures of pa prepores are available1014 , how pa senses low ph , converts to active pore and translocates lf and ef are not well defined without an atomic model of the pa pore . here , by cryo electron microscopy ( cryoem ) with direct electron counting , we have determined the pa pore structure at 2.9- resolution . the structure reveals the long - sought - after catalytic -clamp and the membrane - spanning translocation channel , and supports the brownian ratchet model for protein translocation . comparisons of four structures reveal conformational changes in prepore to pore conversion that support a multi - step mechanism by which low - ph is sensed and the membrane - spanning channel is formed .

METHODS Preparation of PA prepores EM sample preparation and data acquisition Image processing Atomic model building and refinement Extended Data Supplementary Material

PMC3151563

indications for keratoprosthesis ( kpro ) are refractory corneal blindness with repeated corneal graft failure , and monocular or binocular blindness.1 several different devices exist , but the boston type 1 kpro is still the treatment of choice for eyes with adequate tear film and a functional blink.2 surgical preparation of the boston type 1 kpro involves using a full thickness corneal donor button , 8.59.0 mm in diameter , with a central trephined 3 mm hole . centration of the 3 mm kpro pupil on the fovea is integral for image quality . creating the inner 3 mm hole first and then centering the outer ring on the inner hole has been hypothesized to improve centration.3 laser has traditionally been used for refractive surgery with the hypothesis that smoother optical surfaces correlate with improved image quality and visual outcomes.4 mechanical blades create resection by shear force , whereas a femtosecond laser generates pulses that create tissue micro photodisruption and cleavage.4 advances in femtosecond laser software and hardware have allowed for use in customized trephination traditional penetrating keratoplasty ( pkp ) , anterior and posterior lamellar keratoplasty , tunnels for intrastromal ring segments , and astigmatic keratotomy.5 one study of eight corneoscleral rims created a 2.5 mm posterior corneal hole and 7.2 mm diameter stromal pocket , using femtosecond laser , with insertion of an intracorneal alpha cor - like kpro into the stromal pocket.6 in a nonrandomized prospective study , femtosecond top hat pkp versus manual top hat pkp had better visual outcomes , less astigmatism , higher endothelial counts , and a trend toward less rejection.7 sarayba et al demonstrated in a comparative study , with mechanical microkeratome and femtosecond laser , that a smoother stromal bed was created with the femtosecond laser.4 many aspects of corneal surgery can now be done on an automated system with the increased flexibility of laser . tissue separation is more difficult the more edematous the cornea , but can be overcome with higher energy settings.8 from the laser studies , we propose that a smoother vertical cut for the central 3 mm and outer 8.5 mm trephination can be achieved with femtosecond laser . we also propose that maintaining centration of the 3 mm pupil for the boston type 1 kpro will be easier with the femtosecond laser . four human corneoscleral rims donated for research ( utah lions eye bank , salt lake city , ut ) were mounted in an artificial anterior chamber ( altk system , moria / microtech , doylestown , pa ) . the anterior chamber was filled with balanced saline solution ( bss ) ( alcon , fort worth , tx ) running from an intravenous line with the bottle height set to 8 feet to maintain an anterior chamber pressure of approximately 40 mmhg measured using a reichert tonopen applanation tonometer ( depew , erie county , ny ) . two consecutive full - thickness cuts were performed with the femtosecond laser ( 60 khz intralase fs laser , abbott medical optics , abbott park , il ) . the settings for the first cut included an anterior side cut depth of 1200 m , diameter of 3.0 mm , and energy of 2.04 mj . the side cut parameters were as follows : angle 90 , spot separation 3 , and layer separation 3 . for the second cut , the parameters were kept identical , but the diameter was changed to 8.5 mm . the femtosecond laser requires a minimum applanation pressure in order to initiate the laser . for the first cut , this threshold could easily be reached by lowering the laser cone onto the surface of the cornea ( figure 1 ) . given that the laser is not programmed to perform two full - thickness cuts with one treatment card , before the second cut , the laser cone had to be lifted off the surface of the cornea and reapplanated . since the corneas had full thickness trephination , some fluid did egress through the cut , but the corneas maintained the dome configuration . to prevent distortion of the cornea on second applanation , a light touch the cone was then gently applied to the cornea to ensure that the meniscus reached the outer limits of applanation . after the second laser trephination , the laser cone was lifted and the bss line was occluded to prevent fluid flowing through the cut cornea . forceps were used to remove the central 3.0 mm button and then the double - punched donor button from the cornea sclera rim . anterior segment optical coherence tomography ( as - oct ) ( zeiss , jena , germany ) imaging of the donor tissue was obtained by placing the tissue on a flat surface and holding it at the as - oct imaging aperture . separate donor tissue was stored in formalin for light microscopy evaluation with hematoxylin and eosin ( h&e ) staining . the average age of the four donors was 63.2 7.0 years ( range : 5572 ) . the average death to preservation time was 9.1 2.0 hours ( range : 6.610.3 ) . the average central corneal thickness was 552.50 30.60 m ( range : 522595 ) . the average peripheral corneal thickness was 772.0 118.8 m ( range : 650895 ) . there was no difficulty in creating the two separate full - thickness cuts using the femtosecond laser , although egress of fluid from the artificial anterior chamber was noted after creation of the smaller 3.0 mm central trephination , when the cone had to be disengaged and re - engaged . on gross examination , donor tissue was centrally cut from the corneal sclera rim and had a well centered 3 mm button ( figure 2 ) . as - oct imaging of the donor tissue also demonstrated good centration with smooth vertical cuts of the 3 mm button and equal amounts of tissue on either side ( figure 3 ) . as - oct images at 45 , 90 , and 135 also demonstrated tissue with uniform dimensions . light microscopy with h&e staining confirmed smooth - cut edges centrally with a minimum area of coagulative type change at the border of trephination ( figure 4 ) . the use of a femtosecond laser for facilitating manual steps in surgery has gained popularity in ophthalmology . the appeal of using the femtosecond laser comes from its reliability , reproducibility , precision , and speed . clinical use of boston kpro is longstanding , and great success has been achieved with manual trephination . the purpose of this paper is to demonstrate an additional clinical utility with the femtosecond laser . the optics of the boston type 1 kpro relies on centration of the central optic relative to the macula . in creating an ideal optical system , as demonstrated in figures 2 , 3 , and 4 , the femtosecond laser can produce well - centered cuts with smooth trephinated edges . the use of an artificial anterior chamber does require manual placement of the donor tissue , which can still introduce decentralized error . however , once the donor tissue is mounted , there is a central circle in the artificial anterior chamber which permits facile centration of the laser cone . even though the laser can optimize centration in the donor cornea , decentralized error can be introduced if the recipient cornea is not centrally trephinated . it is true that the peripheral edges are more irregular than the central edges ( figure 4 ) . this is most likely due to a compromised anterior chamber system after the full - thickness central cut . currently , the intralase fs system does have the capacity for double cuts under its intralase enabled keratoplasty settings . however , the software only permits for one full - thickness cut , and the second cut can be set to any depth after sparing the anterior 90 m . perhaps optimization of the software to allow two simultaneous full - thickness cuts will decrease irregularity of the peripheral cut and facilitate use of the laser by eliminating the intermediate step of having to disengage and re - engage the applanation cone , and by using only one treatment card for both cuts . while use of the laser does represent a more expensive method for donor tissue preparation , with regards to optimizing the optical system , it permits advancement . we look forward to the clinical use of the technique described in this manuscript and report of any improvements in visual outcome .

we describe a technique for femtosecond laser - assisted preparation of donor tissue for boston type 1 keratoprosthesis to provide accurate double punching of the donor tissue for optimized alignment in the visual axis . the technique was reproducibly performed in four donor corneas mounted in an artificial anterior chamber . this technique can provide optically centered donor tissue with smooth trephinated edges .

Introduction Materials and methods Results Discussion Supplementary material

PMC3136096

a series of obstacles impede the successful delivery of short interfering rna ( sirna ) to the cytosol of target cells [ 1 , 2 ] . strategies involving conjugation of sirna to a wide variety of molecules have been employed to overcome these obstacles . typically , these approaches employ amine- or thiol - modified [ 4 , 5 ] sirna at 3 or 5 ends of one strand of the sirna duplex ( see for review of conjugation chemistry ) . conjugations of sirna to polyethylene glycol , quantum dots containing targeting moieties , cell penetrating peptides such as penetratin , and a range of lipophilic molecules , including cholesterol , have been demonstrated without a loss of silencing ability . recently , we embarked on a project aiming to deliver sirna to the heart using sirna conjugated to the serum protein albumin . the strategy employed involved the use of the common heterobifunctional linker succinimidyl 4-[n - maleimidomethyl]cyclohexane-1-carboxylate ( smcc ) . using this approach , the coupling of smcc to sirna containing an aminohexyl pendant at the 3 end of the sense strand results in an the activated sirna is then covalently linked to serum albumin via reaction with the thiol - containing side chain of cysteine residue 34 , located on the surface of serum albumin molecules ( see figure 1 ) . previous studies have shown that a number of thiol - reactive drugs rapidly and selectively bind to endogenous albumin within a few minutes of administration , due to that fact that free thiol groups are not found on the majority of circulating serum proteins except for albumin . the chemistry used to conjugate the sirna to albumin is not novel ; others have conjugated nanoparticles to sirna using a similar cross - linker , n - succinimidyl-3-(2-pyridyldithio)propionate ( spdp ) . these workers employed a 40 : 1 spdp : sirna ratio to activate the sirna and then gel filtration to remove the unreacted spdp , prior to conjugation . we found significant batch - to - batch variability in the efficiency of the sirna conjugation to bovine serum albumin ( bsa ) using similar conditions and therefore sought to understand the factors limiting the conjugation . here , we report a successful strategy to restore conjugation efficiency , by repeated buffer exchange prior to exposing the amine - modified sirna to smcc . samples of igf - ir sirna used were sourced from dharmacon ( co , usa ) and were modified with an amine for conjugation , with 2-o - methyl modifications for in vivo use , and with a fluorescein label for imaging purposes . each sense strand contained four 2-o - methyl modifications , the sense strand contained a 3 amine modification ( n6 ) and a fluorescein label ( fl ) at its 5 end . the sequence used was as follows , where m refers to 2-o - methyl modification : sense strand : 5-fl(*)gcmcmcaumgugugagamagacc(*)dt(*)dt(*)n6 - 3 , antisense strand : 3-dt(*)dt(*)cggguacacacmucuucumgg-5. ( * ) denotes phosphorothioate linkages . the sirna was dissolved in freshly prepared phosphate - buffered saline ( pbs , ph 7.4 ) or 250 mm borate buffer ( ph 8 or 8.5 ) to a concentration of 0.2 mm and stored at 20c prior to use . 4-[n - maleimidomethyl]cyclohexane-1-carboxylate ( smcc ) was purchased from pierce chemical co ( ca , usa ) and dissolved in dmso ( 9.57 mm ) just prior to use . sirna - bsa covalent conjugates were formed by reacting the 3-amine - modified , fluorescein - labeled sirna with the heterobifunctional linker reagent smcc to form an irreversible amide linkage ( see figure 1 ) . aliquots of sirna and smcc linker were combined to give a ratio of 1 nmol sirna ( 0.2 nm ) to 40 nmol smcc ( 9.58 nm ) in a ~1 : 1 mixture of milliq water and dmso and allowed to react for 1 h at room temperature . excess smcc was then removed by gel filtration chromatography using a nap-5 column ( pharmacia , uk ) , with only the fluorescent flow - through collected . the quantity of sirna collected was then determined by fluorescence using an envision multilabel plate reader ( usa ) with 488 nm excitation/520 nm emission . the activated sirna was then coupled to the surface - exposed cysteine of bsa by incubating the sirna with 2 molar equivalents of bsa for time periods of up to 24 h ( final concentrations of sirna and bsa in the reaction mixture were 30.3 and 60.6 m , resp . ) . the components present in the reaction mixture were examined on a 10% sds - page , together with bsa and sirna standards of known molecular weight ( 66.5 and 14 kda , resp . ) . the gels were loaded with 1 g protein per well and run for 90 min at a constant voltage of 100 v in sds running buffer . sirna was visualized under uv light or using typhoon gel scanner for detection of fluorescein emission at 520 nm , and bsa was visualised following coomassie brilliant blue staining . quantitation of the relative amounts of conjugate and unreacted sirna was performed by densitometric analysis of sirna and bsa bands recorded on gel scans . prior to conjugation , the reconstituted igf - ir sirna was subjected to four rounds of ultrafiltration with pbs buffer using an amicon ultra-4 centrifugal 3 kda filter unit spun at 4000 rpm for 50 min at 4c after each addition of fresh buffer . the concentrated sirna was removed from the filter unit and quantified on an envision multilabel plate reader ( usa ) . as documentation supplied with the commercially supplied amine - modified sirna did not contain any information to suggest the contrary , it was presumed that the sirna would be suitable for conjugation to amine - reactive compounds after simply reconstituting in an appropriate buffer . however , initial attempts to couple the sirna to bsa via the use of smcc , a heterobifunctional cross - linker routinely employed to link amine- to thiol - bearing molecules , met with very limited success . following the literature precedent , sirna was reacted ( activated ) with a 40-fold molar excess of smcc prior to exposure to bsa , as described in section 2 , and at the completion of the reaction samples were analysed via page . figure 2(a ) shows a representative page gel , used to evaluate conjugation efficiency ; scanning under 488 nm excitation reveals sirna standard at 14 kda in lane 3 , with lanes 4 and 5 showing a significant band corresponding to unreacted sirna band and a faint band at the size expected for the sirna - bsa conjugate , approximately 80 kda . densitometric analysis indicated that only 4 - 5% of sirna was conjugated to bsa and that there was no difference in yield between a one - hour and three - hour activation time . whilst there was some batch - to - batch variability , the majority of conjugation reactions performed using several separate batches of amine - modified sirna gave similarly poor results , with less than 20% of sirna in conjugate form . figure 2(b ) shows the same page gel that is shown in figure 2(a ) , in this case after coomassie ( protein ) staining to reveal bsa . there was no clear band apparent at 80 kda , likely indicating that the small amount of sirna - bsa conjugate evident in figure 2(a ) was below the limit of detection using coomassie staining . in troubleshooting the inefficient conjugation reaction , a range of experimental parameters were varied in an attempt to drive the reaction to completion . the reaction was performed overnight , at ph values between 7 and 8.5 , and using a range of sirna , smcc , and bsa concentrations and molar ratios . none of these strategies were successful ( data not shown ) . finally , we hypothesized that there may have been low - molecular - weight species in the sirna as supplied that interfered with the conjugation reaction and , therefore , that extensive ultrafiltration or dialysis prior to activation with smcc to remove these species might improve the outcome of the conjugation reaction . communication with the manufacturer revealed that the sirna was desalted using ammonium acetate ; an unwise choice for amine - modified sirna whose likely purpose is conjugation via the amine functional group , since ammonium ions would become counterions to the polyanionic sirna . ammonia can act as a nucleophile , similar to the primary amine in the modified sirna , and hence is also able to react with the succinimidyl ester group in the smcc linker . figure 3(a ) ( i ) shows the reaction products before ( lane 4 ) and after ( lane 5 ) four rounds of ultrafiltration with pbs . ultrafiltration prior to smcc activation resulted in a 15-fold increase in the proportion of sirna converted to the conjugate form , as evidenced by the band at 80 kda . after coomassie staining of this gel , a clear band at 80 kda was evident in addition to the native bsa band , indicating the bsa component of the conjugate ( figure 3(a ) ( ii ) ) . densitometric analysis of the unreacted and conjugate bsa bands indicated that approximately one third of the bsa was involved in the reaction . this is not unexpected given that approximately 70% of albumin is in the thiol reactive ( mercaptalbumin ) form and that a 2 : 1 albumin : sirna ratio was employed . thus , it appears that removal of the ammonium counterions from the amine - modified sirna prevents the majority of the smcc from being consumed by a competitive side reaction , and thus it allows the reaction of sirna with smcc to proceed more effectively . having demonstrated that the reaction efficiency was dramatically improved by ultrafiltration using pbs , we anticipated that the ratio of smcc linker to sirna used ( 40 : 1 ) was unnecessarily high . figure 4 shows evaluation of the effect of reducing the smcc : sirna ratio on the outcome of the conjugation reaction . there was no reduction in the proportion of sirna in conjugate form upon reducing the smcc : sirna ratio from 40 : 1 to 10 : 1 , and only a modest decrease occurred when the ratio was further reduced to 2 : 1 ( 48% of sirna in conjugate form versus 61% for 40 : 1 ratio , figure 4(a ) ) . thus , it was possible to reduce the smcc concentration during the reaction , with the advantage of reducing the amount of excess linker remaining after the reaction , as well as lowering the cost of synthesis . these data clearly demonstrate that sirna supplied commercially with terminal amine groups can require ultrafiltration or dialysis prior to exposure to linker reagents in order to remove ammonium ions remaining after the desalting step used in the purification process . researchers planning to perform conjugation experiments should consult with the supplier as to the purification conditions , and if it is known or suspected that ammonium - containing salts were used , an ultrafiltration step , as described herein , should be performed prior to use of the sirna .

conjugation of sirna to macromolecules such as serum albumin has multiple potential benefits , including enhanced extravasation via albumin - mediated transcytosis across endothelial cells and reduced renal clearance . in attempting to conjugate sirna to albumin , we used commercially sourced amine - modified sirna and reacted it with the heterobifunctional linker succinimidyl 4-[n - maleimidomethyl]cyclohexane-1-carboxylate ( smcc ) to introduce a maleimide group suitable for conjugation to the thiol group of the surface - exposed cysteine residue ( cys 34 ) within albumin . we found the conjugation of the smcc - treated sirna to bovine serum albumin ( bsa ) to be very inefficient and investigated the cause of the low yield of conjugate . ultrafiltration with phosphate - buffered saline prior to activation with smcc dramatically increased the yield of sirna - albumin conjugate ( ~15-fold ) . communication with the commercial supplier revealed that ammonium acetate buffer was used in a desalting step as part of the sirna purification process prior to supply , likely resulting in ammonium counterions to the sirna polyanion , which would interfere with conjugation by consuming the smcc . after ultrafiltration , a greatly reduced amount of smcc could be used to affect conjugation , without significant reduction in yield . these data indicate that amine - modified sirna sourced commercially may require ultrafiltration or dialysis prior to use in conjugation reactions .

1. Introduction 2. Experimental Methods 3. Results and Discussion 4. Conclusions

PMC2664612

the formation of oxidants is a hallmark of chemical toxicity , inflammation , and other types of environmental stresses . oxidative stress and oxidants also are involved in human diseases that account for significant morbidity and mortality , including cancer , atherosclerosis , and neurodegenerative diseases . although oxidative stress derives fundamentally from the excessive flux of reduced oxygen species , such as superoxide and hydrogen peroxide , secondary products of lipid oxidation may play critical roles in oxidant - associated molecular pathologies . lipid electrophiles such as malondialdehyde , hydroxyalkenals , oxoalkenals , epoxyalkenals , and -ketoaldehydes readily react with proteins , and the formation of covalent adducts is hypothesized to contribute to oxidant - related diseases and toxicities . because they reflect the occurrence of oxidative stress , protein adducts from lipid electrophiles most studies of protein modification by lipid electrophiles have examined a few prototypical lipid oxidation products , such as 4-hydroxynonenal ( hne ) , 4-oxononenal , and -ketoaldehydes ( levuglandins ) using mass spectrometry ( ms ) . this published work has identified relevant protein adduction chemistries with amino acid side chain nucleophiles and in some cases mapped adducts to specific sequences in modified proteins . collectively , the adduct mapping data indicate significant site - specificity in modification , which is further reflected by a recent analysis of the kinetics of competing alkylation reactions of hne with human serum albumin . polyunsaturated fatty acids in living systems are largely present as phospholipid esters and recent studies have focused on novel biological properties of phospholipids bearing epoxyisoprostane and epoxycyclopentenone substituents or a reactive ,-unsaturated carbonyl oxidation product at the sn-2 position . these products stimulate inflammatory signal transduction pathways in vascular endothelial cells , serve as ligands for the scavenger receptor cd36 , and facilitate lipid uptake in macrophages and the formation of foam cells . most of the implicated phospholipids have electrophilic substituents and could be expected to form covalent protein adducts . however , the identities of proteins covalently modified by phospholipid electrophiles have not been reported . a major challenge in studying protein damage by lipid electrophiles is the sheer diversity of products generated . for example , the oxidative decomposition of a phospholipid containing linoleate at the sn-2 position would yield several electrophilic phospholipid products . hne and 4-oxononenal are formed from the -end of linoleate ester , but other reactive electrophiles contain the carboxy - end of the linoleate ester . thus , ketooxododecenoate ( koda ) , hydroxyoxododecenoate ( hoda ) , and other adducts esterified to phospholipid are expected to be among the electrophile adducts formed with proteins from oxidized linoleate - containing phospholipids ( figure 1 ) . characterization of the protein targets of the family of electrophiles formed from a phospholipid is also complicated by the relatively low levels of adducts compared to unmodified proteins in relevant biological systems . analysis of the protein targets of a family of phospholipid electrophiles requires a means of high affinity capture of the adducted proteins . recently , we reported the synthesis of the biotinylated phosphatidylcholine plpbso , which is the biotin - sulfoxide analogue of 1-palmitoyl-2-linoleoylglycerylphosphatidyl - choline ( plpc ) . oxidation of plpbso formed koda , hoda , and other expected linoleate oxidation products and also formed the corresponding electrophile adducts with a model peptide . in that work , we also demonstrated feasibility of streptavidin capture of plpbso - adducted human serum albumin from incubation of the protein with oxidized plpbso . here , we describe the use of plpbso as a probe to identify major protein targets of oxidized phospholipids in human plasma . this approach identified apolipoprotein a1 ( apoa1 ) as the principal target of plpbso oxidation products and led us to map adducts from lipid oxidation products on this protein . we report the identification of adducts from several phospholipid electrophiles , including carboxy - terminal adducts . the concordance of apoa1 site - specific modification between exogenous hne and endogenous phospholipid electrophiles suggests that the identified targets may be preferred modification sites on apoa1 in oxidative stress in vivo . plasma samples were collected from healthy human donors as part of an irb - approved study . 2,2-azobis[2-(2-imidazolin-2-yl)propane]-dihydrochloride ( aiph ) was from wako chemicals ( osaka , japan ) , streptavidin sepharose beads were from ge healthcare ( piscataway , nj ) , and ammonium hydroxide was from sigma ( st . louis , mo ) . apoa1 antibody ( rabbit polyclonal antibody to the rlaeyhakateh peptide of apoa1 ) was from cayman chemical ( ann arbor , mi ) . rabbit polyclonal antibody against hne histidine michael adducts was from calbiochem ( san diego , ca ) . alexa fluor 680-conjugates streptavidin and all secondary antibodies were purchased from molecular probes ( carlsbad , ca ) . mass spectrometry grade trypsin ( trypsin gold ) was from promega ( madison , wi ) . whole blood from fasting , healthy subjects was collected in a 440 ml acd blood collection bag ( baxter ) containing 2 g of dextrose monohydrate , 1.66 g of sodium citrate dihydrate , 188 mg of anhydrous citric acid , 140 mg of monobasic sodium phosphate monohydrate , and 17.3 mg of adenine . plasma was subsequently isolated from red blood cells by centrifuging the whole blood bag at 4200 rpm for 10 min at 22 c . plpbso was dissolved in dmso ( 50 mm ) and was added to plasma at a final concentration 100 m ( < 0.3% v ) . they were placed in 37 c oil bath and stirred for 1.5 h. half of the supplemented and native plasma were aliquoted into eppendorf tubes and stored at 80 c until further use . hdl was isolated by ultracentrifugation ( 1.063 g / ml < d < 1.12 g / ml ) from both native and supplemented plasma . native and plpbso supplemented plasma ( 50 l ) were oxidized with 5 mm aiph overnight at 37 c . a total of 50 l of pbs was added to the sample , and 50 l of protein - g agarose slurry then was used to remove igg side chain that nearly comigrates with apoa1 on sds - page . the supernatant was collected and filtered through amicon 10 kda molecular weight cutoff filter to remove small molecules residual azo initiator and reducing agent . the samples then were diluted to 300 l with 100 mm ammonium bicarbonate , treated with 10 mm dtt at 50 c for 30 min , and then with 20 mm iam in the dark at room temperature . streptavidin sepharose beads ( 100 l slurry ) were washed with 3 500 l of 6 m urea in pbs . the oxidized , reduced , and alkylated native or plpbso supplemented plasma samples were added to the washed streptavidin beads and allowed to rotate for 2 h at room temperature . the supernatant was discarded and the beads were washed with 3 500 l of 6 m urea in pbs with rotation for 15 min at room temperature , then with 3 500 l of 1 m nacl with rotation for 15 min at room temperature , and finally with 3 500 l of 50 mm ammonium bicarbonate . elution of biotinylated proteins was done by selective hydrolysis of the plpbso acyl ester using 100 l of 15% nh4oh in 3 m urea overnight at room temperature . after hydrolysis , the supernatant was collected and the beads were rinsed with 2 100 l of 50 mm ammonium bicarbonate and combined with the eluate , which was placed under house vacuum for 30 min to remove excess nh3 . samples were enzymatically digested for 1824 h at 37 c using 0.5 g of trypsin and the solution then was acidified to ph 3 with formic acid to halt the digestion . then , the samples were evaporated under vacuum , desalted using zip - tips ( millipore c18 , millipore , billerica , ma ) , and resuspended in 200 l of 0.1% formic acid for lc - msms analysis . lc - msms analyses were performed on a thermo ltq linear ion trap instrument ( thermo electron , san jose , ca ) equipped with a thermo surveyor hplc system , nanospray source , and microautosampler . peptides were resolved on a 100 m 11 cm fused silica capillary column ( polymicro technologies , llc , phoenix , az ) packed with 5 m , 300 jupiter c18 ( phenomenex , torrance , ca ) . liquid chromatography was carried out at ambient temperature at a flow rate of 0.2 ml min using a gradient mixture of 0.1% ( v / v ) formic acid in water and 0.1% ( v / v ) formic acid in acetonitrile . peptides eluting from the capillary tip were introduced into the ltq nanoelectrospray source with a capillary voltage of approximately 2 kv . a gas - phase fractionation method was used to increase identifications of lower abundance proteins that were present . each sample was run three times , first with a full scan and precursor selection in the range of m / z 400600 , then m / z 600900 , and finally m / z 9002000 . five data - dependent msms scans were performed on the selected precursor ions from each full scan . msms spectra were recorded using dynamic exclusion of previously analyzed precursors for 30 s with a repeat of 1 and a repeat duration of 2 . msms data were evaluated using the sequest algorithm and the ipi human database , which included both forward and reverse sequences for all entries . sequest outputs were organized and analyzed with a custom database utility called chips ( complete hierarchical integration of protein searches ) . chips allows the user to filter sequest outputs based on sequest output parameters such as xcorr , rsp , and sp scores . in this case , both the native oxidized plasma ( control ) and plpbso supplemented oxidized plasma msms data were filtered using xcorr score thresholds of 2 for 1 + peptides , xcorr 2.5 for 2 + peptides , and xcorr 3 for 3 + peptides with rsp 5 and sp 350 . the false positive rate for peptide identification based on reversed sequence identifications with these criteria was 13.4% . the requirement that protein identifications require at least two distinct peptides per protein reduced the false positive rate for protein identification to 0.4% . proteins that were identified after biotin - streptavidin capture in both the control and plpbso supplemented oxidized plasma sample were removed and the remaining identified proteins were considered covalently adducted by plpbso . proteins captured on streptavidin beads as described above were eluted using 1 m formic acid in acetonitrile / water ( 3:2 , v / v ) overnight at room temperature after washing with the solutions described above . the supernatant was removed and the beads were rinsed with 2 100 l of 50 mm ammonium bicarbonate . the eluted proteins were resolved by 10% nupage novex bis - tris gel and the resolved proteins then were transferred to a pvdf membrane , which was incubated with alexa fluor 680-conjugated streptavidin and imaged using the licor odyssey imaging system ( licor , lincoln , ne ) . both oxidized and unoxidized plasma were rotated with 100 l of protein a - agarose slurry ( see above ) to remove igg light chain , which nearly coelutes with apoa1 . the remaining plasma proteins were resolved on a 10% nupage novex bis - tris gel using mes running buffer and the proteins were electrophoretically transferred to a pvdf membrane . the blocked membrane was incubated with rabbit polyclonal antibody raised to the rlaeyhakateh peptide of apoa1 and immunoblot images were acquired with the licor odyssey imaging system . plpbso supplemented oxidized plasma was incubated with 100 l of streptavidin sepharose slurry followed by the stringent washing as described above . the immobilized biotinylated proteins then were eluted from the beads by hydrolysis of the phospholipid fatty acyl ester bonds with 15% nh4oh for 1215 h at room temperature . the eluted proteins were resolved on a 10% nupage novex bis - tris gel and stained with colloidal blue and the apoa1 band ( 2528 kda ) was excised and subjected to in - gel tryptic digestion . the eluted tryptic peptide solution was evaporated to dryness and the peptides were resuspended in 50 l of 0.1% formic acid in water for lc - msms analysis . msms spectra were acquired as described above , except that gas - phase fractionation was not used ; precursor ions for msms were selected from full scans over the range m / z 3002000 . the data were analyzed with the p - mod algorithm using apoa1 tryptic peptide sequences to detect msms spectra corresponding to mass - modified apoa1 peptides . additional analyses used a similar algorithm in development in our laboratory , called monstermod , which is similar to p - mod , but includes improvements in the scoring and statistical models for generation and evaluation of sequence - to - spectrum matches ( ham , a. l. ; wernke , g. r. ; tabb , d. l. ; kim , h. y. ; liebler , d. c. , manuscript in preparation ) . to characterize the formation of hne adducts in hne - treated samples , isolated hdl was treated with hne at concentrations of 1 m , 10 m , 100 m , and 1 mm . the hne treated hdl proteins were resolved by sds - page on a 10% nupage novex bis - tris gel and stained using colloidal blue followed by tryptic in - gel digestion of the apoa1 band between 2028 kda . to determine hne adducts formed endogenously during hdl oxidation , hdl was incubated with aiph initiator overnight at concentrations of 0 , 100 , 1000 , and 5000 m followed by nabh4 reduction . the hdl proteins then were processed and analyzed as described immediately above . in both cases , the recovered peptides were analyzed by lc - msms and the data then were searched with the p - mod . the use of plpbso as a probe to identify protein targets of phospholipid electrophiles is depicted in figure 1 . oxidation of plpbso with aiph yields reactive aldehydes , ,-unsaturated aldehydes , epoxy-,-unsaturated aldehydes , and other products that can form protein adducts that are stabilized with nabh3cn . oxidation products in which the electrophile comprises a remnant portion of the linoleate chain at the sn-2 position will covalently link the adducted protein to the biotinylated choline headgroup , which enables capture with streptavidin . thus , capture of biotinylated proteins following oxidation enables capture and analysis of the protein targets of plpbso - derived phospholipid electrophiles . we used nabh3cn to stabilize adducts formed with plpbso oxidation products in order to detect both hoda and koda adducts . although nabh4 traps michael adducts more efficiently , it also reduces koda ketones to hydroxyls . on the other hand , the latter reaction was sufficiently slow in our studies to potentially allow redistribution of adducts between michael and imine forms . model studies ( not shown ) with an hne - adducted peptide indicated that reduction with nabh4 for 4 h at room temperature yielded only the michael adduct , whereas reduction with nabh3cn under the same conditions yielded the reduced michael adduct and nonreduced aldehyde and imine adducts . because our product analyses were essentially qualitative in nature although digestion and lc - msms analysis of the captured proteins will enable their identification , the adduct peptides would most likely go undetected because the phospholipid would suppress ionization or induce fragmentations in the phospholipid moiety that would compete with peptide fragmentation , thus , precluding identification by database search of msms spectra . we developed a mild ammonia hydrolysis procedure that cleaves the phospholipid fatty acyl ester bonds with minimal concomitant hydrolysis of the adducted protein ( figure 1 ) . selective ammonia hydrolysis enabled release of the biotinylated proteins from the streptavidin beads and left proteinlipid electrophile adducts containing a carboxy - terminal group ( figure 2 ) . stringent wash of the streptavidin beads containing captured proteins with 6 m urea and 1 m nacl reduced nonspecific binding to the streptavidin . following a tryptic digest of the released proteins , both adducted and base hydrolysis of biotinylated proteins in various concentrations of nh4oh and different temperatures shown in alexa fluor 680-conjugated streptavidin western blot ( left ) and visible coomassie - stained gel ( right ) . disappearance of biotin in the western shows the degree of hydrolysis of the phospholipid headgroup . oxidation of plasma supplemented with plpbso was initiated using the water soluble free radical initiator aiph . the water soluble free radical azo initiator , aiph , was used to initiate a true free radical chain reaction . the use of the azo initiator allows the rate of oxidation to be readily controlled by adjusting the concentration of aiph and the temperature of the reaction . full - scan lc - ms chromatograms of the tryptic digests indicate that the streptavidin beads captured a significant amount of biotinylated protein in the plpbso - supplemented , oxidized plasma , as indicated by the strong ion current in the lc - ms of the corresponding tryptic digests ( figure s1a in supporting information ) . in contrast , very little protein was captured on streptavidin from the nonsupplemented , oxidized plasma samples ( figure s1b in supporting information ) . to identify proteins adducted by plpbso , these tryptic digests then were subjected to lc - msms analysis using a gas - phase fractionation procedure and the data then were searched against the ipi human protein database . the experiment was repeated four times and 21 proteins were confidently identified ( 2 + peptide identifications at 95% confidence ) . the proteins represented by the highest sequence coverage , reflecting those present in the plpbso - labeled protein pool in greatest abundance , are listed in table 1 ( a complete listing of identified proteins is provided in table s2 in supporting information ) . sequence coverage is defined as the fraction of the protein sequence accounted for by msms - identified peptides . to confirm apoa1 adduction using immunoblotting techniques , immunoblotting was done with either a polyclonal antibody raised against apoa1 peptides or alexa fluor 680-conjugated streptavidin . the anti - apoa1 immunoblot indicated that , after oxidation in both the plpbso supplemented plasma and native plasma , the apoa1 band is shifted to higher apparent molecular weight ( figure 3 ) . the alexa fluor 680-conjugated streptavidin immunoblot shows biotin labeling of the higher molecular weight band , which is due to the covalent adduction of phospholipid to the protein . oxidation induces a migration shift of apoa1 in sds - page analysis and streptavidin labels the plpbso - modified apoa1 protein . oxidized plasma was immunoblotted with polyclonal apoa1 antibody followed by alexa fluor 680 antirabbit ( left ) or with antistreptavidin ( right ) . to identify the adducts with carboxy - terminal electrophiles in apoa1 , plpbso - supplemented plasma was oxidized with aiph followed by nacnbh3 reduction to stabilize the adducts and plpbso - adducted proteins were captured with streptavidin beads . after stringent washes to remove nonspecifically bound protein , the beads were treated with 15% ammonium hydroxide to selectively hydrolyze the phospholipid esters . the eluted proteins ( enriched in carboxyl - terminal lipid adducts ) were resolved by sds - page and the apoa1 band was excised for in - gel trypsin digestion followed by lc - msms analysis . analysis of the msms data with the p - mod algorithm searched apoa1 tryptic peptide sequences against the msms data to identify peptides that correspond to apoa1 peptides , including adducts . this approach does not require the user to specify anticipated adduct masses or sequence specificities ( e.g. , his modification ) , but instead performs an unbiased search for spectra that match search sequences with or without mass shifts due to adducts . the program then maps adduct positions based on msms fragmentation and calculates probabilities of random matches . the apoa1 search sequences used included miscleaved peptides that may result from lysine adduction by electrophiles . our criteria for reporting adducts included ( 1 ) presence of b - and / or y - ion series consistent with the adducted peptide sequence , ( 2 ) localization of adduct mass shifts by p - mod to h or k residues capable of reacting with the electrophiles to form the indicated adducts , ( 3 ) presence of diagnostic water loss fragments formed from hydroxy - containing adducts ( e.g. , reduced michael adducts ) . this analysis mapped sites of apoa1 modification and the detected mass shifts enabled identification of the carboxylated electrophiles ( table 2 ) . annotated msms spectra of these adducts are provided in figure 5 and figure s2 in supporting information . the identified residues include both lysine ( k45 , k106 , k188 , k195 ) and histidine residues ( h135 , h155 , h162 ) and included both imine ( schiff base ) and michael adducts with carboxylated simple aldehydes , as well as carboxylated ,-unsaturated aldehydes ( hoda and koda ) . as noted above , the use of nabh3cn as a reducing agent enabled us to distinguish koda and hoda adducts . however , we note that koda adducts corresponding in mass to michael addition products may also be ketoamide adducts , which were recently described by sayre and colleagues and which would not be distinguished by our ms analyses . lc - msms of the t*hlapysdelr tryptic peptide sequence from apoa1 modified with koda ( a ) , with hne ( b ) at his 162 ; of the qkl*helqek tryptic peptide sequence modified with 7-oxoheptanoic acid at his135 ( c ) ; and of the leal*kenggar tryptic peptide sequence modified with 8-oxooctanoic acid at lys182 ( d ) . we note the formation of the apparently novel imine adducts of 7-oxoheptanoic acid , 8-oxooctanoic acid , and 9-oxononanoic acid in these analyses . the latter is formed by the hock cleavage reaction that also generates hne during linoleate oxidation . formation of 8-oxooctanoic acid likely proceeds by fragmentation of the linoleate 9-hydroperoxide to give the 8-yl - octanoic acid . this radical would react with oxygen to give the 8-hydroperoxyoctanoic acid which , upon dehydration , would give the 8-oxooctanoic acid compound . these analyses also identified some electrophile adducts derived from -terminal portion of the phospholipid - bound fatty acyl chain . these detected mass shifts corresponded to schiff base adducts of hexanal , heptanal , octanal , and nonanal ( table s1 in supporting information ) . detection of these species was less consistent from experiment - to - experiment than was detection of the carboxylated adducts described above . this may reflect the fact that the streptavidin capture and wash protocol would select against -terminal adducts , unless apoa1 molecules modified by these species also were covalently labeled with the phospholipid . although adducts were found throughout the protein , the adduction of h162 is of interest because this residue is within a sequence known to interact with lecithin - cholesterol acyltransferase ( lcat ) , an enzyme that contributes to hdl particle maturation . our analyses of the streptavidin - captured apoa1 ( see above ) identified relatively few adducts corresponding to modification by noncarboxylated electrophiles originating from the -terminus of the fatty acid ( e.g. , hne ) . to determine if hne adducts are formed on apoa1 upon oxidation of native hdl associated lipids , hdl particles were isolated from plasma and then oxidized overnight with different concentrations of aiph or treated with hne . anti - hne immunoblotting revealed immunoreactivity of the apoa1 band ( 2026 kda ) from hdl particles oxidized with 5 mm aiph or treated with either 100 m or 1 mm hne ( figure 4a ) . the visible gel in figure 4b shows that a molecular weight shift and band broadening for apoa1 ( similar to that observed in figure 2 , above ) was induced by as little as 100 m aiph , but not by hne treatment . this suggests that the apoa1 migration is affected perhaps by multiple modifications of the apoa1 protein that affect protein charge , shape and/or sds binding , but that hne adduction alone does not produce these effects . isolated hdl treated with hne and oxidized with aiph in different concentrations they are visualized with anti - hne ( left ) or colloidal blue ( right ) . note the migration shift seen in the colloidal blue stained gel in the apoa1 band between 2026 kda is induced by oxidation , but not by hne . hne adduction sites on apoa1 were mapped by lc - msms analysis of in - gel tryptic digests of the apoa1 band from aiph - oxidized and hne - treated samples described above . msms spectra were acquired by data - dependent scanning mode , which automatically samples intact peptide ion signals for msms . the msms spectra were searched against a human protein sequence database with the sequest algorithm with allowance for variable modifications on histidine and lysine residues for borohydride - reduced hne schiff base adducts ( + 140 amu ) or michael adducts ( + 158 amu ) . as a complementary approach , the sequences of apoa1 tryptic peptides were searched against the msms data with the p - mod algorithm to enable detection of spectra corresponding to modified peptides . p - mod hits corresponding to the expected + 140 and + 158 mass shifts on histidine or lysine residues were recorded . all putative spectra of adducts were subjected to manual inspection to verify the quality of sequence matches . ten sites of adduction were identified , including k12 , k23 , k94 , k96 , k107 , h135 , h155 , h162 , h193 , h199 , k208 , and k238 ( table 3 ) . accurate mass measurements for the adduct precursor ions with a thermo ltq - orbitrap instrument confirmed calculated values to within 36 ppm ( table 3 ) . mass error ( ppm ) for measurements of the precursor ions of the observed peptide adducts were analyzed on a thermo ltq - orbitrap instrument and were as follows : k12 , 3.2 ppm ; k23 , 3.8 ppm ; k94 , 3.9 ppm ; k96 , 3.8 ppm ; k107 , 3.7 ; h135 , 4.0 ppm ; h155 , 3.2 ppm ; h162 , 2.9 ppm ; h193 , 3.7 ; h199 , 3.3 ; k208 , 3.8 ppm ; k238 , 5.9 . denotes that the adducts were detected in either 1 of 3 , 2 of 3 , or 3 of 3 independent experiments , respectively . in replicate experiments in which hdl was treated with either hne or oxidized with aiph , histidines in the sequences thlapysdelr and laeyhak were repeatedly found to be modified even with exposure to as little as 1 m hne ( table 3 ) . his162 , which lies within the lcat - interacting domain of apoa1 , was a target for modification by both hne and the carboxyated electrophile koda , representative msms spectra for which are both shown in figure 5 . although lipid electrophiles generated during oxidative stress are widely thought to play critical roles in injury and adaptation to stress , relatively little is known about the protein targets of these species . target identification is complicated by the great diversity of potential targets , the low abundance of modifications , and the difficulty of selectively capturing and analyzing the modified proteins . we have approached this problem by employing affinity - tagged lipid substrates that form oxidation products representative of the same lipid class , but that also enable high affinity capture of the lipid electrophile adducts . the plpbso probe generates oxidation products similar to the corresponding phosphatidylcholine analogue and also forms adducts to proteins . use of plpbso enabled detection of apoa1 as a major protein target of phospholipid electrophiles in plasma . subsequent analysis of apoa1 from oxidized plasma identified novel lipid electrophile adducts derived from phospholipid oxidation products . moreover , adduct mapping studies revealed adduction in a functionally critical region of the protein . apoa1 is the core protein component of hdl particle , which mediates reverse cholesterol transport from tissues to the liver and also exerts antiatherogenic effects . recent work indicates that hdl function is impaired in cardiovascular disease and systemic inflammatory disorders . functional impairment of hdl has been attributed to oxidative modifications of apoa1 by myeloperoxidase ( 4954 ) or reactive products of lipid oxidation . thus , our results indicating that apoa1 is a major target of reactive phospholipid electrophiles led us to focus attention on the types and site - specificity of apoa1 adduction . the biotin tag on the choline headgroup in plpbso allows affinity capture of protein adducts that contain the phospholipid headgroup . these adducts are derived from electrophiles esterified to the phospholipid and form carboxylated adducts upon ammonia hydrolysis . nevertheless , not all adducts identified from streptativin - captured protein are necessarily derived from plpbso . some of the identified adducts may originate from endogenous apoa1 lipid esters and are formed on apoa1 that is also adducted with plpbso . the formation of carboxylated lipid electrophile adducts has been studied to only a limited extent . williams and colleagues characterized koda and hoda adducts formed by decomposition of linoleate hydroperoxide in the presence of purified cytochrome c. we also observed adducts with 7-oxoheptanoic acid and 8-oxooctanoic acid on apoa1 lysine residues . although the formation of these imine adducts is not unexpected , this is the first report of the direct detection of these adducts , to our knowledge . the covalent binding of aldehyde - containing phospholipids has been shown previously to generate antigenic components of oxidized low density lipoprotein ( ldl ) . these changes , in turn , promote recognition of oxidized ldl by c - reactive protein and functional alterations in monocytes exposed to oxidized ldl . a notable characteristic of apoa1 subjected to free radical oxidation is the alteration of its migration characteristics in sds - page ( figure 3 ) . this may reflect the formation of carboxylated adducts on histidine and lysine residues , which would generate a variety of apoa1 forms with modified charge characteristics and possibly altered sds binding . in contrast , treatment with hne does not produce this effect , even when treatment produced comparable levels of hne adduction . our analyses of apoa1 adducts from plpbso - supplemented , oxidized plasma that was captured with streptavidin identified several carboxylated adducts , but relatively few adducts corresponding to electrophiles derived from the -terminus of the fatty acid ( e.g. , hne ) . on the other hand , apoa1 was modified by hne formed during oxidation of isolated hdl with aiph and by exogenously added hne ( table 3 ) . in both cases , the distribution of hne adducts was similar . this confirms that apoa1 can undergo modification both by diffusible hne - like electrophiles and phospholipid - bound electrophiles . the somewhat sporadic detection of hne and other noncarboxylated adducts in plpbso - supplemented plasma following streptavidin capture probably reflects loss of the nonphospoholipid adducted apoa1 proteins during the wash steps to remove nonbiotinylated proteins . this also implies that , under the conditions of our experiments , most apoa1 molecules bear a total of one or two adducts and that these are distributed between all the observed sites . had the protein been more heavily modified , one would have expected to consistently observe hne adducts and other noncarboxylated adducts as well as carboxylated adducts in the streptavidin - captured protein . we identified adducts in two target sequences that have been identified previously in studies of oxidized apoa1 . these sites are immediately adjacent to tyr192 , which was identified by the hazen and heinecke groups as sites of nitration and chlorination in apoa1 . the latter reaction is catalyzed by myeloperoxidase , which binds to this sequence in apoa1 in hdl . we also mapped koda and hne adducts to his162 , which resides within a sequence recently shown to be critical for interaction with lcat . tyr166 undergoes chlorination and nitration in atheromas in vivo and this modification blocks the activation of lcat by apoa1 , which is an obligatory step in hdl particle maturation . his162 is also a solvent - exposed residue in the lcat - activating loop that contains tyr166 ( 44 ) and this raises the possibility that his162 adduction may have functional consequences similar to those reported for tyr166 modifications . our studies demonstrate that his 162 and his193 are among the most easily detected adducts at low hne exposure concentrations in hdl ( table 3 ) . it might be tempting to conclude that this indicates high kinetic reactivity of these residues with electrophiles . however , detection in lc - msms analyses depends not only on relative levels of the adducts , but also on ionization efficiency of the adducted peptides . in a recent study of the kinetics of human albumin adduction at different sites by hne , we noted that the rate constant for formation of the most readily detected adduct ( his67 ) was over 10-fold lower than that for the most reactive site ( his242 ) , which was less readily detected in data - dependent lc - msms analyses . thus , we are planning additional studies of the kinetics of apoa1 modification in plasma and hdl to characterize the hierarchy of reactivity with lipid electrophiles . our results suggest that covalent adducts with lipid electrophiles can mechanistically link lipid peroxidation , oxidative stress and disease mechanisms in a similar manner . in addition , lipid electrophile adducts of apoa1 merit evaluation as potential biomarkers of oxidative stress in vivo .

reactive electrophiles generated by lipid peroxidation are thought to contribute to cardiovascular disease and other oxidative stress - related pathologies by covalently modifying proteins and affecting critical protein functions . the difficulty of capturing and analyzing the relatively small fraction of modified proteins complicates identification of the protein targets of lipid electrophiles . we recently synthesized a biotin - modified linoleoylglycerylphosphatidycholine probe called plpbso ( tallmanet al.chem . res . toxicol.2007 , 7 , 22723417305406 ) , which forms typical linoleate oxidation products and covalent adducts with model peptides and proteins . supplementation of human plasma with plpbso followed by free radical oxidation resulted in covalent adduction of plpbso to plasma proteins , which were isolated with streptavidin and identified by liquid chromatography - tandem mass spectrometry ( lc - msms ) . among the most highly modified proteins was apolipoprotein a1 ( apoa1 ) , which is the core component of high density lipoprotein ( hdl ) . apoa1 phospholipid adduct sites were mapped by lc - msms of tryptic peptides following mild base hydrolysis to release esterified phospholipid adducts . several carboxylated adducts formed from phospholipid - esterified 9,12-dioxo-10(e)-dodecenoic acid ( koda ) , 9-hydroxy , 12-oxo-10(e)-dodecenoic acid ( hoda ) , 7-oxoheptanoic acid , 8-oxooctanoic acid , and 9-oxononanoic acid were identified . free radical oxidations of isolated hdl also generated adducts with 4-hydroxynonenal ( hne ) and other noncarboxylated electrophiles , but these were only sporadically identified in the plpbso - adducted apoa1 , suggesting a low stoichiometry of modification in the phospholipid - adducted protein . both phospholipid electrophiles and hne adducted his162 , which resides in an apoa1 domain involved in the activation of lecithin - cholesterol acyltransferase and maturation of the hdl particle . apoa1 lipid electrophile adducts may affect protein functions and provide useful biomarkers for oxidative stress .

Introduction Experimental Procedures Results Discussion

PMC4867072

elucidating the complex processes that determine the human body 's ability to adapt to specific training stimuli is crucial to improve athletic performance in elite sports . moreover , targeted exercise - based intervention programs provide enormous potential to combat the global epidemic increase of chronic metabolic and inflammatory disorders including obesity , metabolic syndrome , and type ii diabetes . the systemic and physiological responses to exercise are complex and not well understood and involve a wide range of metabolic , immunological , and hormonal changes . hereby , the immediate systemic response to acute exercise is known to be closely dependent on the type , duration , and intensity of exercise . however , also the individuals ' training status has a major impact on the systemic response to acute exercise , indicating an adaptive response to long - term changes in whole - body repetitive exercise stimuli . there is extensive research dealing with the specific response of different exercise regimes in relation of age , gender , training status , and physical conditions including obesity and diverse chronic inflammatory disorders [ 39 ] . most of these studies focus on exercise - induced responses of selected plasma cytokines altered by acute exercise stimuli [ 1014 ] . besides their roles in mediating immunological responses , specific cytokines have recently emerged to profoundly influence diverse nonimmunological processes , including metabolic functions ( e.g. , glucose and lipid metabolism ) , repair and/or prevention of tissue damage , and skeletal muscle remodeling [ 1517 ] . these diverse actions depend on complex and still largely undeciphered crosstalk with various other metabolic and humoral biomolecules that are also released in response to physical activity . these signaling molecules can act synergistically as well as in an independent manner to meet the specific physiological and metabolic demands of the exercising body . the simultaneous detection of a wide range of plasma biomolecules by recently developed multianalyte profiling methods now offers the possibility of generating a more comprehensive picture of the complex systemic responses to exercise . in the present study , an evaluation of at least 90 plasma proteins in trained and untrained individuals at rest and in response to a standardized endurance exercise protocol was conducted . this approach offers the unique opportunity to obtain more comprehensive insights into the systemic response to acute exercise under the precondition of different training statuses . our analyzed panel included various cytokines and their soluble receptors , as well as acute - phase proteins , hormones , metabolic marker proteins , and muscle damage markers . we hypothesized that regular endurance exercise should be reflected by specific baseline plasma signatures and that acute exercise - provoked plasma variable profiles should display training status dependent response patterns . the determination of protein profiles under different preconditions will contribute to a better understanding of the complex mechanisms of systemic exercise - induced adaptation processes . these findings can be of importance for training monitoring in elite sports and in the development of exercise - based strategies for the prevention and/or therapy of chronic metabolic and inflammatory degenerative diseases . in a previous study , we analyzed skeletal muscle specimen from a subset of these individuals using an off - gel liquid chromatography tandem mass spectrometry ( lc - ms / ms ) approach . proteome profiling revealed differences in various metabolic , especially mitochondrial proteins between endurance - trained and untrained individuals under resting conditions and in response to acute exercise . in addition , a subgroup analysis of participants and variables was performed in connection with extracellular dna traps and cell - free dna evaluation in plasma . healthy male endurance - exercise - trained ( eet ) athletes as well as untrained / sedentary ( sed ) individuals were recruited based on training history and aerobic capacity . the inclusion criteria for the eet group ( n = 19 ) were regular endurance exercise training of minimum 5 hours per week for at least 5 years before participation and an individual maximal oxygen uptake ( vo2max ) > 57 ml / min / kg body weight ( bw ) . the inclusion criteria for the sed group ( n = 17 ) were less than 2 hours of unspecific sports per week and a vo2max < 47 ml / min / kg bw . all experimental procedures were approved by the local ethics committee of justus - liebig university giessen , the university hospital of tuebingen , and the university of ulm . each participant underwent a medical screening and had to sign a written consent before the experimental procedure . initially , maximum oxygen uptake was determined by cardiopulmonary exercise testing ( metalyzer ; cortex biophysics , leipzig , germany ) with an incremental protocol on a bicycle ergometer ( excalibur sport ; lode , groningen , netherlands ) to determine the vo2max including capillary blood lactate diagnostics ( biosen ; ekf diagnostics , magdeburg , germany ) to evaluate the individual performance . within one to four weeks after the performance diagnostic , the main exercise protocol was performed . after an overnight fast ( from 21.00 pm to 08.00 am ) , participants received standardized breakfast and lunch on the day of acute exercise . on the day before , blood samples were taken at rest ( called the the exercise setup comprised an acute endurance exercise protocol on a bicycle ergometer for 50 min at an intensity ( watts ) corresponding to 80% of their individual vo2max . a 10 min warm - up at 60% of their individual vo2max preceded the endurance trial . study participants were instructed to drink a defined amount of water corresponding to their body weight at two points in time during the cycling exercise protocol . at ten minutes and at three hours after the acute exercise protocol , further blood samples were collected ( called post and 3 h , resp . ) . edta blood samples were collected for whole blood cell count and preparation of plasma samples . complete blood count was accomplished by synlab lab services ( synlab medical care center kassel gmbh , kassel , germany ) . edta tubes for plasma isolation were kept on ice and centrifuged at 1600 g for 10 min within 30 min after collection ( universal 320r , hettich centrifuges , tuttlingen , germany ) . plasma supernatant was collected , immediately frozen in liquid nitrogen , and stored at 80c . quantification of 90 analytes in edta plasma samples was performed at myriad rules - based medicine ( myriad rbm , austin , usa ) using humanmap v. 1.6 of their luminex - based multiplex immunoassays as described by the vendor . only variables that could be detected in at least 80% of the plasma samples were taken into account for further statistical analysis , using a procedure for multiplex measurements [ 21 , 22 ] . this procedure resulted in the inclusion of 78 of the 90 available variables of humanmap v. 1.6 . samples of the included variables that had no detectable value were set at half of the lowest measured value for the corresponding variable . to exclude biased measures due to plasma volume shift , changes in plasma concentrations were calculated from haemoglobin and haematocrit values , and concentrations of postexercise variables were corrected as described by kraemer and brown and accomplished previously . in addition , granulocyte - colony stimulating factor ( g - csf ) was chosen for separate analysis on the basis of recent findings in its role in exercise delayed neutrophil apoptosis and progenitor cell mobilization [ 14 , 24 ] . g - csf concentration in plasma samples was measured using a high sensitive enzyme - linked immunosorbent assay ( hselisa ) kit ( r&d systems , minneapolis , usa ) according to the manufacturers ' instructions . the concentration of g - csf was calculated using mikrowin 2000 v. 4.34 ( mikrotek labor systems , overath , germany ) . mean standard deviation ( sd ) and median with the 1 . and 3 . quartile for all included variables can be found in supplemental table s1 in supplementary material available online at http://dx.doi.org/10.1155/2016/4851935 . physiological data were statistically analyzed using wilcoxon 's rank sum test for independent samples for differences between groups and presented as mean sd and median with the 1 . and 3 . plasma and whole blood data were statistically analyzed using a nonparametric two - factorial analysis of variance ( anova ) for longitudinal data to assess the main effects of time and group and their interaction effects . the chosen nonparametric method allows us to analyze the data without the need of normality or data transformations . the analysis is based on the so - called relative treatment effects ( rtes ) and data are presented as rte with the corresponding 95% confidence intervals ( ci ) . briefly described , a rte has a value between 0 and 1 and indicates the probability that a measurement in one group at a given time point is larger than a value of this variable in any other combination of group and time . keeping this in mind , a rte > 0.5 means that the eet group tends to have larger values than the sed group , whereas a rte < 0.5 must be interpreted as tendency towards lower values in eet group . for more details when a significant main effect was observed , appropriate post hoc pairwise comparison for different time points ( repeated nonparametric one - factorial anova for longitudinal data with time as factor ) or between groups ( wilcoxon 's rank sum test ) was performed . correlation analysis in the eet group for plasma variables that differ significantly at baseline between groups with their corresponding vo2max was performed using spearman 's rank correlation . for all analysis a p value < 0.05 was considered to be significant . whenever necessary to account for multiple testing , holm 's correction method for p values was performed . in addition , the scores of the area under the curve with respect to the increase ( auci ) were calculated for all plasma variables and compared by student 's t - test or wilcoxon 's rank sum test when appropriate as described previously . the statistics software r , version 3.0.2 , and the packages nparld and nparcomp , graphpad prism 5.0 ( graphpad software , la jolla , usa ) , jmp pro 10.0.2 ( sas institute , cary , usa ) , and ibm spss v. 21 ( ibm corp . , armonk , usa ) were used for statistical analysis . the acute endurance exercise at 80% vo2max in watts led to a significantly higher workload in the eet group ( p < 0.001 ) . there were no differences in lactate levels and heart rate ( hr ) between the two groups during the one - hour - lasting exercise ( table 1 ) . lactate values started to increase significantly after half of the main exercise protocol was completed and remained elevated until the end of the exercise trial , with no significant differences between groups ( p < 0.001 ) . total leukocytes and neutrophil numbers significantly increased over time until 3 h after exercise . lymphocytes and monocytes increased directly after exercise . at three hours after exercise , lymphocytes had decreased to baseline , whereas monocytes remained elevated ( table 2 ) . twenty - nine of the 78 included variables showed an effect in response to the acute exercise intervention without differences between the eet and sed group ( table 3(a ) ) . marked responses to exercise were observed for the proinflammatory and chemoattractant interleukins interleukin- ( il- ) 1 beta ( il-1 ) and il-16 directly after exercise , whereas the anti - inflammatory il-1 antagonist il-1 receptor antagonist ( il-1ra ) showed a delayed increase 3 h after exercise . matrix metalloproteinase-9 ( mmp-9 ) also increased over time , whereas its antagonist , the tissue inhibitor of metalloproteinase-1 ( timp-1 ) , showed an immediate response pattern and declined to baseline values 3 h after exercise . other inflammation - associated analytes , including the neutrophil - derived extracellular newly identified receptor for advanced glycation end - products binding protein ( en - rage ) as well as myeloperoxidase ( mpo ) and the chemokine epithelial - derived neutrophil - activating protein 78 ( ena-78 ) and the t - cell - specific protein rantes ( rantes ) , also increased markedly in response to exercise and remained elevated at the 3 h time point . we could further observe increased levels of growth factors such as brain - derived neurotrophic factor ( bdnf ) , granulocyte - colony stimulating factor ( g - csf ) , stem cell factor ( scf ) , and vascular endothelial growth factor ( vegf ) directly after exercise . g - csf , scf , and vefg remained elevated 3 h after exercise in contrast to bdnf . the same pattern applies for plasma levels of adhesive glycoproteins thrombospondin 1 and von willebrand factor ( vwf ) which also increased immediately after exercise and remained elevated . the rtes of all 29 variables that display a significant time effect are clustered in figure 2(a ) . seven plasma variables differed between groups at baseline but were not affected by the exercise intervention ( table 3(b ) ) . in the eet group , elevated levels could be observed for the protease inhibitor protein alpha-2-macroglobulin ( a2macro ) , apolipoprotein ( apo ) a - i ( apo a - i ) , a major component of high - density lipoproteins ( hdl ) , and t - helper ( th ) type 2 and hematopoietic cytokine il-5 . in contrast , significant higher baseline values in the sed group were evident for the very low density lipoprotein component apo c - iii , the chemokine macrophage - derived chemokine ( mdc ) , the acute - phase protein serum amyloid p - component ( sap ) , and the adipokine leptin . fifteen of the analyzed variables showed significant main effects for both group and time ( table 3(c ) ) . we found group differences for the chemoattractant proteins il-8 and monocyte chemotactic protein-1 ( mcp-1 ) with higher levels in the eet group . both chemokines increased directly after exercise in both groups while mcp-1 already displayed elevated baseline levels in the eet group . likewise , baseline values of the anti - inflammatory il-10 differed between groups with increased levels in the eet group . in response to exercise cytokine il-10 increased in both the eet and sed group but remained elevated only in the eet group 3 h after exercise . th2-type cytokines il-3 and il-13 revealed no post hoc time effects but differed markedly between groups with higher levels in the eet group . exercise - provoked increases in tumor necrosis factor - alpha ( tnf- ) levels could only be observed in the eet group directly in response to exercise . concordantly , plasma levels of tumor necrosis factor receptor 2 ( tnfr2 ) were significantly higher in the eet group already at rest and an immediate increase in response to exercise could only be observed in the eet group . the protease inhibitor protein alpha-1-antitrypsin ( aat ) was elevated in the eet group compared to the sed group with exercise dependent time effects in the sed group 3 h after exercise . we further found myoglobin , an early and sensitive marker for muscle membrane damage , to be elevated in the eet group at rest . an exercise - provoked release over time could be observed in both groups , with increasing levels at 3 h after exercise . similarly , the insulin antagonist anabolic growth hormone ( gh ) differed between groups with significantly elevated levels at rest in the eet group . gh immediately increase in response to exercise and remained elevated 3 h after exercise in both groups . in contrast , elevated levels of insulin could be detected in the sed group compared to the eet group . insulin dropped immediately after exercise in both the eet and sed group but remained decreased at 3 h after exercise only in the eet group . nine of the analyzed variables exhibited a significant interaction effect between group and time ( table 3(d ) ) . extracellular matrix proteins mmp-2 and mmp-3 revealed different time curves between eet and sed group . an effect of exercise for mmp-2 could only be observed in the eet group and mmp-3 remained elevated only in the eet group at 3 h after exercise . the increase of il-6 was more pronounced in the eet group immediately after exercise but post hoc tests revealed no significant differences between groups ( p = 0.07 ) . interaction effects could also be observed for the muscle damage markers creatine kinase mb ( ck - mb ) and heart - type fatty acid - binding protein ( fabp ) . fabp increased directly after exercise with higher levels in the eet group and remained elevated in both groups . ck - mb was significantly higher at all three sampling points in the eet group . however , a delayed increase also became apparent in the sed group at 3 h after exercise . the nonparametric analysis revealed no significant main or interaction effects of time and groups for 19 variables . rtes for significant variables with group , group and time , and interaction ( group by time ) effect for eet and sed groups ( tables 3(b)3(d ) ) are clustered in figure 2(b ) . plasma variables that differ significantly between the eet and sed group at baseline ( pre ) are displayed in table 4 . correlation analyses of baseline plasma variable values with vo2max for the eet group revealed a significant positive correlation for apo a - i ( r = 0.66 , p = 0.03 ) and a negative correlation for leptin ( r = 0.68 , p = 0.02 ) . in the present study , we compared the plasma protein profiles of endurance - exercise - trained ( eet ) and sedentary ( sed ) individuals at rest and the effects of an acute bout of intensive endurance exercise on these circulating blood variables over time in both groups . the systemic response to acute exercise relies on coordinated crosstalk of body tissues to maintain metabolic and immune homeostasis [ 3234 ] . the immune system has to preserve an adequate immune function towards exogenous pathogens while maintaining self - tolerance to endogenous components that are released during exercise from skeletal muscle and other tissues . the acute endurance exercise increased circulating levels of muscle cell damage and permeability markers such as myoglobin , ck - mb , and fabp illustrating the need for an adequate self - tolerance in response to strenuous exercise . here , an increase in muscle damage markers could be observed , especially in the eet group . key cytokines il-3 , il-5 , il-7 , and il-13 , which have the ability to skew t - cell cytokine secretion to t - helper ( th ) type 2 responses , stimulate dendritic cell differentiation , and maintain t - cell homeostasis [ 35 , 36 ] , showed significant group and time effects with higher levels in the eet group . these effects could also be observed for immunosuppressive mediators il-10 and tnfr2 [ 37 , 38 ] . these findings indicate that regular exercise provokes acute as well as long - term changes in th2-type ( il-5 , il-6 , and il-13 ) and regulatory t - cell ( treg ) ( il-10 ) cytokines with more pronounced effects in trained athletes compared to sedentary participants . despite their role in inflammation immune function and metabolic regulation are tightly interconnected when it comes to exercise - induced alterations . high il-10 levels , for example , have been reported to promote insulin sensitivity in humans and protect against insulin resistance by diminishing tnf mediated intracellular signaling in adipocytes . apo a - i , a major component of high - density lipoprotein ( hdl ) particles , has been shown to have anti - inflammatory effects on blood cells and adipocytes but is also postulated to improve cellular energy glucose metabolism and increase glucose uptake by muscle cells . here , we could observe a positive correlation between apo a - i and the endurance capacity represented by vo2max in the eet group under resting conditions . apo a - i was not affected by acute exercise but showed a significant group effect with higher levels in the eet group . in line with this , the lc - ms / ms skeletal muscle analysis revealed elevated levels of key enzymes of the oxidative energy metabolism in the endurance - trained muscle and , therefore , the ability for enhanced muscular glucose utilization . during intense endurance exercise , glucose is the major fuel for the exercising muscle mobilized from muscle and liver glycogen stores . the observed decrease in blood insulin in response to exercise , especially in the eet group , enables the mobilization of glucose for energy expenditure . one key player in the acute metabolic signaling is the pleiotropic cytokine il-6 that is locally secreted by the contracting skeletal muscle and found to be elevated in circulation in response to exercise . in the present study , the highest values of il-6 for both groups were measured immediately after exercise with a more pronounced effect in the eet group . il-6 has been demonstrated to provide metabolic effects , like enhanced glycogenolysis and fatty acid oxidation , which correspond to the elevated abundance of mitochondrial enzymes of the tricarboxylic acid and oxidative phosphorylation in the endurance - trained skeletal muscle and , therefore , the possibility of utilizing more substrate for energy expenditure . il-6 knockout mice showed an impaired carbohydrate and lipid metabolism and , in addition , were insensitive towards the metabolic effects of leptin despite elevated circulating plasma leptin level . our study revealed significantly lower plasma leptin levels in the eet group but no impact of acute exercise . in addition , within the endurance - trained individuals , leptin was negatively correlated with the vo2max at baseline . a reduction in blood leptin levels has recently been implicated with both an improved endurance training status and changes in body composition . in addition to its metabolic functions , leptin is considered to have proinflammatory activity and stimulates th1-type cytokines whereas the lower leptin levels in the eet group are in line with the abovementioned shift towards th2-type cytokines in trained individuals . in contrast , the proinflammatory cytokine tnf- and the chemokines il-8 and mcp-1 revealed group and time effects and were also increased in the eet group after exercise . in the context of exercise remarkably , the protease inhibitors aat and a2macro were found to be elevated in the eet group whereas aat also increased 3 h after exercise in the sed group . aat serves as an inhibitor of neutrophil - derived elastase and might reflect a response to exercise - provoked neutrophilia and increased elastase release to limit its adverse effects . similarly , a2macro , a well - known inhibitor of proteinases , released by neutrophils in the inflammatory process , protects human tissue from collateral damage caused by unregulated proteolytic activity . moreover , a2macro binds to several cytokines , including tnf- , il-1 , and il-6 , and modulates their biological activity . the tnf- complex may be removed by complex formation with a2macro from circulation and the biological activity of il-1 is inhibited by a2macro . in contrast , il-6 activity has been reported to remain partly active , and il-6 is protected from degradation and available for its target cells when binding to a2macro . supportive evidence comes from the regulation of mmp-2 and mmp-3 in response to exercise with significantly higher levels in the eet group . despite their role in the degradation of extracellular matrix proteins and tissue remodeling , mmps influence inflammatory processes through their functional inactivation of cytokines and chemokines like il-1 [ 51 , 52 ] . therefore , protease inhibitors and mmps might have the potential to diminish an excessive proinflammatory reaction in response to exercise . accordingly , we suggest that the trained organism initiates long - term adaptive processes to cope with exercise - induced inflammatory and metabolic stress , thus allowing performances at higher intensities without adverse effects . a deeper understanding of exercise - induced alterations offers the possibility of prescribing customized exercise as medicine in metabolic and inflammatory disorders like obesity , metabolic syndrome , and type ii diabetes . in the present study , we could demonstrate that acute strenuous endurance exercise has differential effects on circulating blood variables , depending on the athlete 's training status . while a wide range of variables responded simultaneously to exercise in sedentary and endurance - trained individuals , we also identified plasma proteins that differ between groups in response to acute exercise . in addition , the analysis revealed plasma proteins that were not affected by acute exercise but differed between groups at baseline , indicating long - term homeostatic adaptations caused by regular endurance exercise training . as a consequence of the systemic and cellular adaptation to inflammatory and metabolic stress evoked by regular exercise , trained athletes are capable of performing at absolute higher workloads . to achieve a similar systemic burden in both groups , a standardized relative workload protocol was chosen ( indicated by similar lactate and hr values ) that allows a physiological comparison between groups . in consequence , the eet group performed at a higher absolute workload and it should be considered that postexercise plasma levels might also be affected by differences in exercise intensity of both groups . we believe that our analysis provides meaningful new insights into the effects of endurance exercise on metabolic and inflammatory signaling and homeostasis . systemic blood variables that are capable of monitoring exercise adaptation are of scientific value not only to improve athletic performance in elite sports but also to develop exercise - based intervention programs in the prevention and treatment of chronic metabolic and inflammatory diseases .

acute physical exercise and repeated exercise stimuli affect whole - body metabolic and immunologic homeostasis . the aim of this study was to determine plasma protein profiles of trained ( eet , n = 19 ) and untrained ( sed , n = 17 ) individuals at rest and in response to an acute bout of endurance exercise . participants completed a bicycle exercise test at an intensity corresponding to 80% of their vo2max . plasma samples were taken before , directly after , and three hours after exercise and analyzed using multiplex immunoassays . seventy - eight plasma variables were included in the final analysis . twenty - nine variables displayed significant acute exercise effects in both groups . seven proteins differed between groups , without being affected by acute exercise . among these a2macro and il-5 were higher in eet individuals while leptin showed elevated levels in sed individuals . fifteen variables revealed group and time differences with elevated levels for il-3 , il-7 , il-10 , and tnfr2 in eet individuals . an interaction effect could be observed for nine variables including il-6 , mmp-2 , mmp-3 , and muscle damage markers . the proteins that differ between groups indicate a long - term exercise effect on plasma protein concentrations . these findings might be of importance in the development of exercise - based strategies in the prevention and therapy of chronic metabolic and inflammatory diseases and for training monitoring .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Concluding Remarks

PMC3981203

in ano - rectum different types of neoplastic as well non - neoplastic lesions can occur . among the latter , hemorrhoids , anal fissure or polyps are common . this is a report of an anal growth , which was clinically diagnosed as thrombosed pile but histopathology and immunohistochemistry revealed its real nature . a 60-year - old male presented to surgical out - patients department complaints of bleeding from rectum and pain during defecation . he reported a history of weight loss and anorexia , and at examination he was pale and cachectic . on rectal examination a growth about 321 cm was found in the rectum , 4.3 cm from the anal verge . the clinical diagnosis was of a thrombosed pile , therefore it was excised and sent for histopathological examination . the examination at the microscopy revealed a tumor replacing the entire wall thickness with ulceration of overlying epithelium . the tumor cells were disposed in fascicles and sheets ( figure 1a ) and were spindle - shaped . few of them showed prominent inclusion like nucleoli and atypical mitotic figures , while many contained blackish pigment ( figure 1b ) . the tumour cells were strongly positive for s-100 and hmb-45 ( figure 1d ) . based on these findings a diagnosis of sarcomatous variant of malignant melanoma figure 1microphotograph of the tumor in the ano - rectum showing ( a ) the tumor cells arranged in long parallel running fascicles ( b ) the high - power view of individual cells with prominent spindling and abundant blackish pigment in many of them ( c ) schmorl 's stain demonstrating the pigment to be melanin stained as blue green granules and ( d ) strong positivity for hmb-45 in the tumor tissue . microphotograph of the tumor in the ano - rectum showing ( a ) the tumor cells arranged in long parallel running fascicles ( b ) the high - power view of individual cells with prominent spindling and abundant blackish pigment in many of them ( c ) schmorl 's stain demonstrating the pigment to be melanin stained as blue green granules and ( d ) strong positivity for hmb-45 in the tumor tissue . thereafter , an abdomino - perineal resection with removal of bilateral inguinal , pelvic and mesorectal lymph nodes was carried out . on histological examination no residual tumor was identified at the primary site , however one of the inguinal lymph nodes was found to harbor the metastatic tumor deposits . the patient received radiation and chemotherapy and was then discharged and advised for a regular follow - up . the patient however came back after 13 months and on computerized tomography ( ct ) scan of abdomen showed multiple secondaries in liver . the patient was discharged on his own request and thereafter no follow up could be obtained . malignant melanoma is a rare neoplasm in the ano - rectum accounting for only 13% of all tumors . most of the malignant melanomas are of classical type and the sarcomatous variant , although it is well known in skin has been rarely reported at this site . by reporting this case we intended to alert clinicians and pathologist about its occurrence and importance of its recognition . a dermal sarcomatoid melanoma has even worse prognosis than classical malignant melanoma with a 5-year survival rate accounting to only 15% , despite an aggressive , multimodality approach . it has been reported that 38% of patients have already metastatic disease at the time of diagnosis . however , its prognosis in ano - rectum is not yet clear due to its rarity at this site . the present case also showed a dismal prognosis , as patient developed disseminated malignancy despite aggressive therapy . further data on the various prognostic factors is needed , which is possible only if more cases are reported . its diagnosis also may be difficult even on histology and it may be confused with other more common spindle cell tumors i.e. leiomyoma , haemangioma , haemangiopericytoma , neurilemmoma , neurofibroma , granular cell tumor , spindle cell lipoma , gastrointestinal stromal tumor , malignant fibrous histiocytoma , leiomyosarcoma , rhabdomyosarcoma , fibrosarcoma and spindle cell carcinoma . the key diagnostic feature is the melanin production , which may be absent in approximately 20% of the cases . in such difficult cases only immunostaining can help in achieving the correct diagnosis . a sarcomatous melanoma in ano - rectum is seldom reported in the literature and the present case highlights several key points . most importantly , when faced with a spindle cell lesion of anal canal , especially in an elderly , the diagnosis of sarcomatous melanoma must be included in the differential . as occurred in our case , the clinical impression is often misleading , therefore it is important that the pathologist maintain a high index of suspicion . the histologic appearance of sarcomatous melanoma mimics other spindle cell tumors , therefore immunohistochemical stains play an important role in diagnosis . since present therapeutic strategies may not necessarily alter the prognosis , an early recognition of this unusual variant of malignant melanoma is a key to prolong the survival .

we report a case of extremely rare variant of ano - rectum malignant tumor . the tumor is often misdiagnosed as either hemorrhoids or rectal polyp , which are benign diseases . on histology also this variant may be confused with other more commonly occurring spindle cell lesions in this area ; its recognition is therefore important as it normally has a poor prognosis .

Introduction Case Report Discussion

PMC3461772

the closure of the mesenteric defect following bowel resection has traditionally been undertaken , especially in open surgery , in an attempt to prevent bowel herniation and subsequent strangulation . with the advent of laparoscopic surgery however , the value of this practice has been challenged and many surgeons do not routinely close the resultant mesenteric defect following laparoscopic bowel resection . proponents of the former approach point to the risk of small bowel herniation through an open mesenteric defect , potentially leading to bowel obstruction and rarely to catastrophic mesenteric ischemia . in support of this argument , several case studies have reported complications attributed to the nonclosure of the mesenteric defect ; interestingly , these studies often relate to laparoscopy - assisted colostomies.[25 ] conversely , opponents of the mesenteric defect closure advocate that such practice may result in the constriction of the bowel mesentery , inadvertent ligation of blood vessels and/or mesenteric hematoma formation and could , therefore , compromise the blood supply to the bowel anastomosis and lead to anastomotic dehiscence . here we propose a simple technique , applicable to both open and laparoscopy - assisted colectomies , that enables a quick closure of the mesenteric defect while minimizing the risk of blood vessel injury . following bowel resection , the ligatures used for mesenteric vessel ligation are left long [ figure 1 ] . once the bowel anastomosis is completed , the long ligatures on either side of the mesenteric defect are tied together closing the defect [ figure 2 ] . following bowel resection , mesenteric vessels are ligated and the ligatures are left long long ligatures on either side of the mesenteric defect are tied together closing the defect unfortunately , there is paucity of high - quality data to inform surgical practice on this topic with much of the available evidence based on case reports and nonrandomized , observational studies . this report describes a simple technique that enables the closure of the mesenteric defect while minimizing the risk of inadvertent damage to the blood vessels supplying the bowel anastomosis . our mesenteric defect closure technique represents an alternative to those previously reported in the literature , such as stapled closure or closure using a continuous running suture , and is currently being evaluated in a prospective study undertaken in our unit .

the closure of the mesenteric defect following bowel resection remains controversial . proponents of the intervention cite the risk of bowel herniation through an open mesenteric defect and subsequent bowel obstruction whereas supporters of the opposing view advocate that such practice may lead to inadvertent compromise of the bowel blood supply . we describe a novel technique that enables efficient mesenteric defect closure while minimizing the risk of blood vessel injury .

INTRODUCTION TECHNIQUE DISCUSSION

PMC4706602

culturomics was recently introduced in our laboratory ( urmite , marseille , france ) as an alternative method to expand the human gut repertoire , thanks to the multiplication of culture conditions with a rapid identification method by matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) . consequently , new genera and species of bacteria are found thanks to this technique , , , , . the characterization of new bacteria was previously based on a combination of phylogenetic and genotypic characteristics , including 16s rrna sequence similarity , gc percentage and dna - dna hybridization , , , . unfortunately , significant limits such as their threshold values , which were not applicable to all species or genera , did not promote their utilization . the advent of high - throughput sequencing techniques made many bacterial genome sequences available in public databases . our laboratory recently developed a taxonogenomic approach based on a systematic comparison of phenotypic ( especially the maldi - tof spectrum ) characteristics and genomic characteristics with the phylogenetically closest bacteria found in the databases , , , , . here we present an analysis of the characteristics that allowed us to describe gabonia massiliensis strain gm3 , a bacterium isolated from a stool specimen from a healthy gabonese male youth and classified into the porphyromonadaceae family , created in 2011 by krieg . we describe the classification , biochemical features and complete genomic sequencing and annotation of g. massiliensis gen . , sp . nov . strain gm3 (= csur p1909 = dsm 100571 ) . the stool sample was collected in lbamba ( gabon ) in january 2015 after approval from the national ethics committee of gabon ( registration 0023/2013/sg / cne ) and ifr48 ( marseille , france ) ( registration 09 - 022 ) and after receipt of the patient 's signed consent . the specimen was taken from a healthy 16-year - old gabonese male youth ( body mass index , 19.03 kg / m ) . he was a member of the nzebi tribe , which is one of the biggest tribes in gabon . the stool sample was stored at 80c until it was sent to urmite ( marseille , france ) . in april 2015 , the stool sample was diluted in phosphate - buffered saline ( life technologies , carlsbad , ca , usa ) . obtained inoculum was preincubated anaerobically at 37c in an anaerobic blood culture bottle ( biomrieux , marcy ltoile , france ) enriched with blood and rumen . after the preincubation stage , 100 l was incubated for 24 to 48 hours on 5% sheep 's blood enriched columbia agar ( biomrieux ) at 37c under anaerobic conditions using an anaerobic atmosphere generator system ( genbag anaer ; biomrieux ) . enriched columbia agar solid medium ( biomrieux ) and identified by maldi - tof and 16s rrna sequencing . maldi - tof identification consisted of picking one isolated bacterial colony with a pipette tip from a culture agar plate and spreading it as a thin film on an mtp 384 maldi - tof target plate ( bruker daltonics , leipzig , germany ) , , , , . then 2 l of matrix solution ( saturated solution of -cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid ) was overlaid on each smear and allowed to dry for 5 minutes . measurements and maldi - tof analysis were conducted as previously described , , , , . identification depended on the score obtained ; a score of 2 with a validly published species enabled identification at the species level ; a score of 1.7 but < 2 enabled identification at the genus level ; and a score of < 1.7 did not enable any identification , , , , . if the colonies corresponded to a new bacterium , confirmation of the identification had to occur with a 16s rrna pcr coupled with sequencing . that was performed using geneamp pcr system 2720 thermal cyclers ( applied biosystems , bedford , ma , usa ) and abi prism 3130xl genetic analyzer capillary sequencer ( applied biosystems ) , respectively . if the confirmation was positive , the spectrum of the new bacterium was entered into the bruker database . the 16s rrna nucleotide sequence was corrected using chromas pro 1.34 software ( technelysium , tewantin , australia ) , and the blastn searches were performed by the national center for biotechnology information tool ( http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/blast.cgi ) . for phylogeny , sequences were aligned using clustalw , and phylogenetic inferences were obtained using the neighbour - joining method within mega software . numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 1000 times to generate a majority consensus tree . the 16s rrna for g. massiliensis strain gm3 was deposited in genbank under accession number ln849789 . strain gm3 was cultivated on 5% sheep 's blood enriched colombia agar ( biomrieux ) in anaerobic conditions at different temperatures ( 28 , 37 , 45 and 55c ) to assess its range of growth temperatures . the genbag anaer and genbag microaer systems ( biomrieux ) were respectively used to assess the ability of the bacterium to grow anaerobically or in microaerophilic conditions at 37c , using the same medium culture . for halophilia and ph testing , four nacl concentrations ( 0 , 5 , 15 and 45% ) and three different phs ( 5 , 7 and 8.5 ) were tested . api gallery systems were performed with api zym , api 20 a and api 50ch ( biomrieux ) for biochemical assays . oxidase ( becton dickinson , le pont de claix , france ) and catalase assays ( biomrieux ) were done separately . sporulation was tested by performing thermal shock on bacterial colonies ( diluted in phosphate - buffered saline ) at 80c for 10 minutes . the motility of strain gm3 was tested by observing its fresh colony between blades and slats using a dm1000 photonic microscope ( leica microsystems , nanterre , france ) with a 40 objective lens . the antibiotic susceptibility of g. massiliensis was tested using sirscan discs of amoxicillin , doxycycline , rifampicin , nitrofurantoin , vancomycin , amoxicillin / clavulanic acid , clindamycin , gentamicin , imipenem , erythromycin , metronidazole , trimethoprim / sulfamethoxazole , amikacin , ciprofloxacin and tobramycin antibiotics ( i2a , montpellier , france ) . g. massiliensis strain gm3 was observed after negative coloration using a tecnai g20 ( fei , limeil - brevannes , france ) transmission electron microscope at an operating voltage of 60 kv . this coloration was observed by using the dm1000 photonic microscope ( leica microsystems ) with a 100 oil - immersion objective lens . using the mate - pair strategy , the genomic dna ( gdna ) of g. massiliensis gm3 was sequenced on the miseq sequencer ( illumina , san diego , ca , usa ) , , , , . the gdna was barcoded in order to be mixed with 11 other projects with the nextera mate - pair sample prep kit ( illumina ) . the mate - pair library was prepared with 1 g of gdna using the nextera mate - pair illumina guide , and the gdna sample was simultaneously fragmented and tagged with a mate - pair junction adapter . the pattern of fragmentation was validated on an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) with a dna 7500 labchip . the dna fragments ranged in size from 1 to 10 kb , with an optimal size of 4.08 kb . no size selection was performed , and only 464 ng of tagmented fragments were circularized , , , , . the circularized dna was mechanically sheared to small fragments with an optimal size of 569 bp in microtubes on the covaris s2 device ( covaris , woburn , ma , usa ) . the library profile was visualized on a high sensitivity bioanalyzer labchip ( agilent technologies ) , and the final library concentration was measured at 24.4 after a denaturation step and dilution at 15 pm , the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell . an automated cluster generation and sequencing run were performed in a single 39-hour run on 2 251 bp . a total of 10.1 gb of information was obtained from a 1189k / mm cluster density with a cluster passing quality control filters of 99.1% ( 22 579 000 clusters ) . the reads obtained were trimmed and then assembled using the clc genomics wb4 software , , , , . using prodigal ( http://prodigal.ornl.gov/ ) with default parameters , we predicted open reading frames ( orfs ) . however , if they spanned a sequencing gap region , the predicted orfs were excluded . the predicted bacterial protein sequences were searched against the genbank and clusters of orthologous groups ( cogs ) databases using blastp . both trnas and rrnas signal peptides and the number of transmembrane helices were predicted using signalp and tmhmm , respectively . orfans were identified when their blastp e value was lower than 1e-03 for an alignment length greater than 80 amino acids . if alignment lengths were smaller than 80 amino acids , we used an e value of 1e-05 . artemis and a dnaplotter were used for data management and visualization of genomic features , respectively . finally , we used the mauve alignment tool ( version 2.3.1 ) for multiple genomic sequence alignments . the mean level of nucleotide sequence similarity at the genome level between g. massiliensis and other bacteria was estimated using the average genomic identity of orthologous gene sequences ( agios ) homemade software . overall , this software is combined with other software : proteinortho ( to detect orthologous proteins between genomes compared two by two , and then retrieves the corresponding genes ) and the needleman - wunsch global alignment algorithm ( to determine the mean percentage of nucleotide sequence identity among orthologous orfs ) . annotation and comparison analyses were performed using the multiagent software system dagobah that includes figenix . g. massiliensis strain gm3 was compared with bacteroides salanitronis strain dsm 18170 , alistipes shahii strain wal 8301 , alistipes finegoldii strain dsm 17242 , barnesiella viscericola strain dsm 18177 , ornithobacterium rhinotracheale strain ort - umn 88 , bacteroides fragilis strain ych46 , bacteroides dorei strain hs1 and helicobacter pylori strain 26695 . the spectrum resulting from the 12 clean g. massiliensis spots did not identify bacteria because there was no spectrum match with those of bruker database . using 16s rrna sequence , strain gm3 , exhibited 91% identification with barnesiella viscericola ( ab267809.1 ) ( fig . 1 ) . the similarity of 16s rrna in these two bacteria led to classification of this new bacterium into the porphyromonadaceae family , created in 2011 by krieg ( table 1 ) . this 16s rrna nucleotide similarity to barnesiella viscericola was lower than the threshold of 95% recommended by stackebrandt and ebers to delineate a new genus . consequently , g. massiliensis gen . the spectrum resulting from g. massiliensis was thus added into the bruker database ( fig . 2 ) , and a gel view was performed in order to see the spectra differences between other close bacteria ( fig . 3 ) . then the 16s rrna sequence of g. massiliensis was deposited in genbank under accession number ln849789 . , strain gm3 (= csur p1909 = dsm 100571 ) , is a gram - negative bacillus ( fig . 4 ) . strain gm3 is a non motile , non - spore - forming , oxidase - negative but catalase - positive bacterium . growth was observed from 28 to 45c , with optimal growth at 37c , and colonies were obtained after 48 hours of culture . g. massiliensis grew in salinity conditions of 0 and at a ph of 7 . it exhibited translucent colonies with a diameter of 1.5 mm on 5% sheep 's blood enriched colombia agar . strain gm3 is a preferentially anaerobic bacillus but was able to grow in microaerophilic atmosphere and unable to grow in aerobic conditions . individual cells had a mean diameter of 0.5 m and a length of 1.25 m when observed with electron microscopy ( fig . 5 ) . using api 20a , we observed positive reactions with d - glucose , d - lactose , d - maltose , d - cellobiose , d - mannose , d - raffinose , d - rhamnose and d - trehalose but not with d - mannitol , d - sucrose , salicin , d - xylose , l - arabinose , glycerol , d - melezitose and d - sorbitol . negative reactions were recorded for indole formation , urease and gelatin using the same gallery . using api 50ch , we concluded that g. massiliensis is able to ferment inositol , methyl-d - mannopyranoside , methyl-d - glucopyranoside , n - acetylglucosamine , esculin ferric citrate , d - cellobiose , d - maltose , d - lactose , d - sucrose , inulin , starch , d - arabitol and potassium-5-ketogluconate . results from the api zym gallery showed enzymatic activities of alkaline phosphatase , esterase ( c4 ) , esterase lipase ( c8 ) , acid phosphatase , naphthol - as - bi - phosphohydrolase , - and -galactosidase and n - acetyl--glucosaminidase . antibiotic susceptibility testing revealed that g. massiliensis strain gm3 is sensitive to nitrofurantoin , doxycycline , rifampicin , amoxicillin / clavulanic acid , and imipenem . however , it remained resistant against amoxicillin , vancomycin , clindamycin , gentamicin , erythromycin / metronidazole , tobramycin , trimethoprim / sulfamethoxazole , amikacin and ciprofloxacin . the main phenotypic and biochemical characteristics of g. massiliensis compared to the closest bacteria are summarized in table 2 . it was composed of 19 scaffolds ( composed of 66 contigs ) ( fig . the 3,288 predicted genes , 3,227 were protein - coding genes and 61 were rna genes ( two genes were 5s rrna , two genes were 16s rrna , two genes were 23s rrna and 55 genes were trna genes ) . a total of 1,807 genes ( 56.00% ) were assigned as putative function ( by cogs or by nr blast ) , and 113 genes ( 3.50% ) were identified as orfans ( table 3 ) . the remaining genes were annotated as encoding hypothetical proteins ( 1,229 genes , 38.08% ) . nucleotide content and gene count levels of the genome are summarized in table 3 , while the distribution of genes into cogs functional categories is presented in table 4 . the draft genome of gabonia massiliensis strain gm3 was larger in size ( 4.26 mb ) than bacteroides salanitronis strain dsm 18170 , alistipes shahii strain wal 8301 , alistipes finegoldii strain dsm 17242 , barnesiella viscericola strain dsm 18177 and ornithobacterium rhinotracheale strain ort - umn 88 ( 4.24 , 3.76 , 3.73 , 3.08 and 2.4 mb , respectively ) but smaller than bacteroides fragilis strain ych46 and bacteroides dorei strain hs1 ( 5.28 and 5.24 mb ) . the g+c content ( 37.9% ) of gabonia massiliensis strain gm3 is larger than that of ornithobacterium rhinotracheale strain ort - umn 88 ( 37.2% ) but smaller than those of alistipes shahii strain wal 8301 , alistipes finegoldii strain dsm 17242 , barnesiella viscericola strain dsm 18177 , bacteroides fragilis strain ych46 and bacteroides dorei strain hs1 ( 57.2 , 56.6 , 51.7 , 46.6 , 43.3 and 41.6 , respectively ) . the gene content ( 3228 ) of gabonia massiliensis strain gm3 is larger compared to those of ornithobacterium rhinotracheale strain ort - umn 88 , barnesiella viscericola strain dsm 18177 , alistipes shahii strain wal 8301 and alistipes finegoldii strain dsm 17242 ( 2,289 , 2,557 , 3,090 and 3,226 ) but smaller than that of bacteroides fragilis strain ych46 , bacteroides dorei strain hs1 and bacteroides salanitronis strain dsm 18170 ( 4,670 , 4,024 and 3,543 ) . similarly , the number of protein - coding genes ( 3,227 ) of gabonia massiliensis strain gm3 were higher than those of ornithobacterium rhinotracheale strain ort - umn 88 , barnesiella viscericola strain dsm 18177 , alistipes shahii strain wal 8301 and alistipes finegoldii strain dsm 17242 ( 2,210 , 2,464 , 2,996 and 3,108 ) but lower than those of bacteroides fragilis strain ych46 , bacteroides dorei strain hs1 and bacteroides salanitronis strain dsm 18170 ( 4,578 , 4,024 and 3,543 ) . however , the distribution of genes into cogs categories was similar in all genomes compared ( fig . in addition , gabonia massiliensis strain gm3 shared 3,231 , 3,132 , 4,269 , 4,373 , 3,686 , 2,499 , 1,579 and 2,312 orthologous genes with alistipes finegoldii strain dsm 17242 , alistipes shahii strain wal 8301 , bacteroides dorei strain hs1 , bacteroides fragilis strain ych46 , bacteroides salanitronis strain dsm 18170 , barnesiella viscericola strain dsm 18177 , helicobacter pylori strain 26695 and ornithobacterium rhinotracheale strain ort - umn 88 ( table 5 ) . although gabonia massiliensis is a draft genome , making the comparison complex , we propose a summary of the comparison of genome content of gabonia massiliensis and other bacteria in table 6 . among species with standing in nomenclature , agios values ranged from 83.25 between alistipes shahii strain wal 8301 and alistipes finegoldii strain dsm 17242 to 56.41 between gabonia massiliensis strain gm3 and alistipes finegoldii strain dsm 17242 ( table 5 ) . to evaluate genomic similarity among the studied strains , we determined two parameters , agios ( table 5 ) , which was designed to be independent from dna - dna hybridization ( ddh ) , and digital ddh , which exhibits a high correlation with ddh , ( table 7 ) . on the basis of phenotypic , phylogenetic and genomic analyses , we formally propose the creation of gabonia massiliensis gen . this bacterium was isolated from a stool sample from a healthy gabonese male youth in marseille , france . n. gabonia , the latin name of gabon , the sub - saharan african country where the stool specimen was collected ) . this is a gram - negative , non - spore - forming and non motile bacillus . it showed positive activity for alkaline phosphatase , esterase ( c4 ) , esterase lipase ( c8 ) , acid phosphatase , naphthol - as - bi - phosphohydrolase , - and -galactosidase and n - acetyl--glucosaminidase . massiliensis , of massilia , the latin name of marseille , where g. massiliensis was isolated ) . individual cells exhibited a diameter of 0.5 m and a length of 1.25 m . it is a gram - negative , non - spore - forming and nonmotile bacillus . g. massiliensis showed positive reactions for alkaline phosphatase , esterase ( c4 ) , esterase lipase ( c8 ) , acid phosphatase , naphthol - as - bi - phosphohydrolase , - and -galactosidase and n - acetyl--glucosaminidase . it was sensitive for nitrofurantoin , doxycycline , rifampicin , amoxicillin / clavulanic acid and imipenem . g. massiliensis type strain gm3 (= csur p1909 = dsm 100571 ) was isolated from a stool sample of a healthy gabonese male youth . its 16s rrna sequence was deposited in genbank under accession number ln849789 , and the whole genome shotgun sequence has been deposited in genbank under accession number cypv00000000 .

culturomics coupled with taxonogenomics is currently used to isolate and characterize new bacteria . here we describe the features and complete genome sequence of gabonia massiliensis strain gm3 , an anaerobic gram negative , non - spore - forming and catalase - positive bacillus isolated from a stool specimen of a healthy gabonese male youth . belonging to a new genus called gabonia , it exhibits a genome of 4 261 752 bp including 37.9% gc content and 3,288 predicted genes .

Introduction Material and Methods Results Conclusion Taxonomic and nomenclatural proposals

PMC4868229

one of the most popular athletic games , soccer , requires a basic element of strong physical power almost at the threshold level1 , through which successful athletic performance is marked by technical and tactical activities2 . physical power is based on muscle strength and durability , quick reaction and agility , and flexibility , and it involves complex components , such as the relationship between the strength of the quadriceps femoris and anaerobic power3 . to maintain balance and allow for quick reactions and agility during soccer , the iterative interaction among the joints of the lower limb has a significant effect on the players athletic performance4 . the muscle strength of the knee joint plays a role in supporting the body s weight . during a game , however , it also supports the center of body mass while performing such dynamic movements as kicking the ball , jumping , and changing direction , which all rely on quick reaction , agility , and coordination5 . functional abnormalities of the knee joint can include overuse , repetitive injuries , or unstable proprioception due to repetition of limited movements within the joint s mobility range6 . to maintain the stability of the knee , it is important to evaluate the level of the muscle strength balance , to assess endurance using isokinetic exercises , and then to facilitate muscle development accordingly7 . in soccer , the foot and ankle joints have the highest rates of injury , and functional movement impairment can be a cause of instability through insufficient muscle strength and lack of proprioception8 . even when structural instability is excluded , functional instability can cause the weakening of the ankle joint when movement patterns are repetitive , direction is abruptly changed9 , or active movement is carried out intensely10 . in competitive elite sports like soccer , these problems can manifest themselves as chronic issues such as a higher risk of injury or functional abnormalities11 . the coordinated activity of the joints and muscles involved in movements of the ankle joint further enhances the functional stability by improving stable weight support as well as motor control function12 . through the measurement of isokinetic variables , the aim of this study was to assess the impacts of functional and chronic asymmetry on the muscle strength of the left and right knee and ankle joints in soccer players at a range of athletic performance levels , and to generate useful data for the development of effective functional exercise programs for symmetrical muscle strength . the 49 study subjects were recruited from 3 different levels of athletic performance : professional soccer players ( k league , n=15 ) , amateur players ( k3 league , n=16 ) , and college soccer players ( u league , n=18 ) . all of the participants provided written consent to participate in the study , after the study procedure and methods were explained to them . table 1table 1.general characteristics of participantsvariablesnpp ( k league)ap ( k3 league)up ( u league)age ( yrs)1526.94.421.11.920.31.5height ( cm)16179.47.3175.54.1180.94.6weight ( kg)1877.58.070.44.676.66.1values are meansd . pp : professional players , ap : amateur players , up : university players shows the general characteristics of the subjects . all the subjects understood the purpose of this study and provided their written informed consent prior to participation , in accordance with the ethical standards of the declaration of helsinki . pp : professional players , ap : amateur players , up : university players the isokinetic muscle functions of the knee and ankle joints were the main experimental variables of the study , and they were measured using the humac norm testing and rehabilitation system ( csmi medical & solution , usa ) . subjects were seated in the measurement chair , and the torque of the knee joint was aligned with the rotating axis of the dynamometer by adjusting the position with a table - tube cross clamp and a pedestal column clamp . the thigh area and the upper body were tightly fixed using a strap and a belt so that the quadriceps exercise would not be affected by external force during the flexion and extension exercises of the knee joint . additionally , to isolate the muscle strength of the area of interest , the ankle was fixed with a strap by adjusting the length of the lower leg and the adjustment axis with an adapter , after which the flexion and extension exercises of the knee were performed . the range of motion was determined by measuring the maximum flexion from the position in which the joint was extended ( 0 ) in a sitting position . care was taken so that the joint would not be either hyperextended more than 0 or hyperflexed more than 135. measurements were taken by group . as a warm - up exercise , the subjects performed flexion and extension exercises of the lower limb 3 times below maximum and 1 time at maximum , at angle speeds of 60/sec and 180/sec . then , the measurements were collected 5 times at the angle speed of 60/sec and 15 times at the angle speed of 180/sec . after the measurements were obtained at each speed , the subjects were told to repeat the flexion and extension of the knee joint , while resting for approximately 2 minutes in a sitting position . next , the subjects were seated in the measurement chair in the same manner described above for the knee joint , and the strength of the plantar flexion and dorsiflexion muscles in the ankle were measured at angle speeds of 30/sec and 120/sec . after completion of a warm - up exercise of plantar flexion and dorsiflexion 3 times below maximum and 1 time at maximum , at the angle speed of 30/sec and 120/sec , measurements were taken 5 times at 30/sec and 15 times at 120/sec . after the performance was measured at each speed , the subjects repeated the flexion and extension exercises , while resting for approximately 2 minutes in a sitting position . for each measurement , all of the data analysis was conducted using spss for windows version 20.0 ( spss inc . , usa ) . means and standard deviations were computed for all the measurement items . to test group differences , one - way anova was performed , and when significant differences were found , post - hoc comparisons were performed using the scheffe method . tables 2table 2.comparison isokinetic peak torque of the knee jointppapupextensor ( nm)60/secright207.823.2205.318.8254.134.0left202.733.1204.920.3246.943.6180/secright123.021.1118.629.3148.121.3left128.027.9120.022.4147.320.8flexor ( nm)60/secright133.628.8129.120.0152.038.1left131.525.2128.117.5145.125.2180/secright90.125.3111.919.295.424.3left91.922.5104.411.997.621.2values are meansd , * p<0.05 , * * p<0.01 , * * * p<0.001 . pp : professional players , ap : amateur players , up : university players and 3table 3.comparison isokinetic ratio of the knee jointppapup60/sec ( % ) right64.110.964.111.560.013.2left64.97.064.910.559.27.6180/sec ( % ) right72.613.564.111.566.220.3left71.810.764.910.566.713.9values are meansd . pp : professional players , ap : amateur players , up : university players show the results of the one - way anova on peak isokinetic strength and differences in muscle strength of the knee and ankle joints , and the level of muscle strength on the left and the right , according to level of athletic performance . peak extensor strength of the knee joint was significantly higher in the university players ( up ) than the professional players ( pp ) or amateur players ( ap ) , both in the right and left knee at the angle speed of 60/sec ( both p<0.001 ) and the angle speed of 180/sec ( both p<0.01 ) . the peak flexor strength of the knee joint was significantly higher in ap than pp or up , at the angle speed of 180/sec in the right knee ( p<0.05 ) . values are meansd , * p<0.05 , * * p<0.01 , * * * p<0.001 . pp : professional players , ap : amateur players , up : university players values are meansd . pp : professional players , ap : amateur players , up : university players tables 4table 4.comparison isokinetic peak torque of the ankle jointppapupplantar flexion ( nm)30/secright106.828.3125.025.8109.128.1left101.820.9109.221.2105.423.9120/secright51.718.880.823.462.920.8left56.820.076.134.758.717.0dorsi flexion ( nm)30/secright30.38.734.95.822.35.1left29.97.433.65.535.45.1120/secright16.27.019.64.29.92.2left17.25.918.43.717.22.6values are meansd , * p<0.05 , * * p<0.01 , * * * p<0.001 . pp : professional players , ap : amateur players , up : university playersand 5table 5.comparison isokinetic ratio of the ankle jointppapup30/sec ( % ) right28.54.028.77.021.14.6left30.18.331.77.835.610.3120/sec ( % ) right32.69.726.29.417.76.3left32.311.627.09.530.29.1values are meansd , * * * p<0.001 . pp : professional players , ap : amateur players , up : university players show the results of the one - way anova on the peak strength of the isokinetic function in the ankle joint , as well as the ratio of muscle strength on the left and right , according to participant s level of athletic performance . the peak plantar flexor strength of the ankle joint was higher in ap than pp or up at the angle speed of 120/sec in the right ankle ( p<0.01 ) . the peak dorsiflexor strength was significantly lower in up than pp or ap in the right ankle at the angle speed of 30/sec , and at the angle speed of 120/sec ( p<0.001 ) , and significantly higher in the left ankle at the angle speed of 30/sec ( p<0.05 ) . up showed a significant asymmetry in muscle strength between the left and right sides compared to pp and ap at the angle speeds of 30/sec and 120/sec ( p<0.001 ) . the ratio of the muscle strength of the ankle joint in the left and the right was significantly lower for up than pp or ap in the right ankle at the angle speed of 30/sec and at the angle speed of 120/sec ( p<0.001 ) . values are meansd , * p<0.05 , * * p<0.01 , * * * p<0.001 . pp : professional players , ap : amateur players , up : university players values are meansd , * * * p<0.001 . reinforcing muscle strength to maintain the stability of the knee joint is essential for soccer players13 . deficiencies in quadriceps muscle strength have a direct link to the risk of injury and can cause osteoarthritis in the knee joint14 . it has been reported that injury frequency increases in athletes when there is a difference of 10% or more between the strength of the left and the right quadriceps femoris15 , 16 . one cause of instability of the main joints in the lower limbs is instability of flexion and extension movements , accompanied by loss of muscle strength ; this instability can be manifested in a variety of forms17 . in the present study , up had significantly higher isokinetic strength of the quadriceps femoris than pp or ap , and no asymmetry was observed in the left and the right muscle strength . however , a previous study on the isokinetic strength of elite players and ap demonstrated that the muscle strength rate of the hamstring was higher in elite players18 . although in that study it was observed , as in this study , that up had significantly higher quadriceps femoris strength , pp showed balance between the quadriceps femoris and the hamstring and a higher muscle strength rate of the hamstring . it is thought that the muscle strength rate of the hamstring is higher because it enhances the stability of the knee joint to increase speed over short distances and to allow for precise foot techniques . the muscle strength in the ankles of soccer players also differs depending on players athletic performance level . a study that compared the isokinetic muscle strength in the ankle joint of up and pp found that it was greater in pp , who were at a higher level of athletic performance than up19 . in this study , the plantar flexor strength was higher in ap compared to pp and up , and no asymmetry in muscle strength in the left and the right was observed , whereas the dorsiflexor strength was significantly lower in up compared to pp and ap . our finding is similar to that of a previous study that reported that muscle strength imbalance in the foot joints , deficiencies in proprioception , mechanical instability , and weakening of the peroneal tendon , which can all be caused by repetitive training , overuse , and damaged soft tissue , are interrelated20 . thus , it is believed that the tibialis anterior , which is involved in dorsiflexion and inversion , is relatively weak in muscle strength expression , in comparison to the strength of the soleus , gastrocnemius , and peroneal tendon that are involved in plantar flexion and eversion . muscle balance is an indicator employed to judge the level of athletic performance , and the extent of muscle imbalance due to exercise and incorrect positioning is one factor that influences performance21,22,23 . soccer is a sport in which a variety of connecting movements , including running , abrupt stopping , changing direction , jumping and landing , kicking and passing , repeatedly occur and these movements require complex functioning of the knee and ankle joints . the hamstring and the calf muscle work to maintain the stabilized center of body mass or to push the ground surface using reaction force , and the quadriceps femoris and dorsiflexors are used when moving forward , kicking , or adjusting a ball by flexion of the hip joint , extension of the knee , and dorsiflexion of the ankle . it is not only the balance between the agonist and antagonist muscles , but also the balance between the left and the right , and between the proximal joint and distal joint in a weight - bearing position that are involved during exercise24 , 25 . as seen in the up in this study , the weakening of dorsiflexors due to muscle strength asymmetry in the right ankle joint is thought to cause the strengthening of the extension muscles of the knee joint in order to maintain the center of body movement and stability . to maintain stability and balance in lower limb movement , it is essential to maintain muscle activation26 . when a movement is made by a soccer the player , the muscles in the foot are already activated to function before the foot touches the ground27 , and when the knee joint sustains a large shock like landing or when the center of mass moves , the contraction greatly increases in the quadriceps femoris28 . it is thought that muscle strength asymmetry in the ankle joint may strengthen the muscles of the knee joint as a counterbalance in order to maintain the center of body mass . in the future , to investigate the extent of muscle activation due to asymmetry between left and right , studies must be conducted on muscle contraction as the center moves with the use of kinetic variables .

[ purpose ] the aim of this study was to collect basic data on the effect of asymmetry on the muscle strength of the left and right knee and ankle joints of soccer players at varying athletic performance levels , to guide the development of improved exercise programs . [ subjects and methods ] forty - nine soccer players at three athletic performance levels participated : 15 professional , 16 amateur , and 18 college . knee extensor and flexor strength were measured at 60/sec and 180/sec , and ankle plantar flexor and dorsiflexor strength were measured at 30/sec and at 120/sec . variables were analyzed by one - way anova . [ results ] college soccer players showed greater muscle strength at 60/sec and 180/sec in the knee extension muscles of both the right and the left sides , lower muscle strength at 30/sec and 120/sec in the dorsiflexor of the right ankle , and similar levels of asymmetry between left and right . the maximum muscle strength on the same side significantly differed in the right ankle joint , with asymmetry between left and right at 30/sec and 120/sec . [ conclusion ] these findings suggest that muscle strength asymmetry in the ankle joint may lead to counterbalancing muscle strengthening of the knee joint to maintain the center of body mass .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC5266791

interstrand crosslinks ( icls ) are toxic dna lesions that prevent the separation of the two filaments in the dna double helix , impairing dna replication and transcription . the fanconi anemia ( fa)/brca pathway is the major interstrand crosslink repair process in higher eukaryotes ( knipscheer et al . , 2009 , kottemann and smogorzewska , 2013 ) . activation of this pathway depends on the specific mono - ubiquitination of the fanci and fancd2 proteins by the fa core complex ( ceccaldi et al . , 2016 , garcia - higuera et al . , 2001 , mutations in the fa genes cause a rare genetic disorder characterized by hypersensitivity to crosslinking agents , chromosomal instability , and cancer predisposition ( deans and west , 2011 ) . despite the enormous effort of several research groups , a mechanistic understanding of the activation of the fa icl repair pathway is only starting to emerge ( walden and deans , 2014 ) . a key player in the recognition of icls is the fa anchor complex ( consisting of the fancm dna translocase , faap24 , and two histone - like factors named mhf1 - 2 ) , which , in turn , recruits the fa core complex to the dna damage site ( ciccia et al . , 2007 , coulthard et al . , 2013 , kim et al . , 2008 , yan et al . , 2010 ) . the core complex is composed of three distinct protein assemblies with non - redundant functions : fancb - fancl - faap100 ( hereinafter called bl100 ) , fancc - fance - fancf ( cef ) , and fanca - fancg - faap20 ( ag20 ) ( huang et al . , 2014 , rajendra et al . , 2014 ) . while the ag20 and cef play loosely defined ancillary roles ( wang and smogorzewska , 2015 ) , the bl100 complex is a three - member ubiquitin ligase , with fancl as the minimal unit required for ubiquitination ( huang et al . , 2014 , fancl is an e3 ligase containing a ring domain that is essential for the interaction with the e2 enzyme , ube2t / fanct , and for the mono - ubiquitination of the fanci and fancd2 molecular targets ( cole et al . , 2010 , hodson et al . , 2011 , hodson et al . , 2014 , machida et al although fancl is active in isolation , its catalytic function is greatly stimulated by the interaction with fancb and faap100 ( huang et al . an activating conformational switch in the fancl catalytic center , or whether fancb and faap100 play an architectural role important for mono - ubiquitination , is unknown . multiple other architectural aspects of the mono - ubiquitination of fanci and fancd2 likewise remain poorly understood . first , crystallographic studies on mouse proteins indicate that the fanci - fancd2 ( id2 ) hetero - dimer forms a pseudo-2-fold symmetric arrangement . however , the mono - ubiquitination sites map at the dimerization interface within the id2 complex ( joo et al . , 2011 ) , suggesting that a reconfiguration of this interface is needed at some point during the mono - ubiquitination reaction to allow for access of the e3 ligase assembly . second , whether a stable id2 complex is recruited to the dna damage site prior to mono - ubiquitination is unclear , although recent cryoelectron microscopy ( cryo - em ) studies on the unmodified human proteins support this notion ( liang et al . , 2016 ) . remarkably the symmetric hetero - dimer captured by crystallography can not be recognized in the electron microscopy ( em ) structure , suggesting that the id2 complex might visit different conformational states throughout the ubiquitination reaction . finally , although both members of the id2 dimer need to be mono - ubiquitinated for activation of the icl repair pathway ( sims et al . , 2007 , smogorzewska et al . , 2007 , timmers et al . , 2001 ) , so far , biochemical reconstitution has mainly focused on the minimal ability of the bl100 e3 ligase to mono - ubiquitinate fancd2 , with little to no fanci mono - ubiquitination observed ( rajendra et al.,2014 ) . how equivalent mono - ubiquitination of both fanci and fancd2 is achieved and whether any of the ancillary proteins in the fa core complex are involved in this process remain unresolved questions . to address these issues , we have used em , combined with crosslinking and mass spectrometry ( xl / ms ) , to analyze the architecture of the bl100 complex and its substrate id2 , either in isolation or with other components of the fa core complex . we found that bl100 forms a symmetric dimer of trimers , inviting a molecular model for the equivalent ubiquitination of the dimeric id2 assembly . the cef component of the fa core complex binds to the two extremities in the bl100 dimer , probably acting as a molecular matchmaker that mediates the bl100 and id2 interaction . we also uncovered an unexpected structural function for cef , which stabilizes the dimerization interface of the id2 complex . we postulate that this function might have a direct role in the efficient mono - ubiquitination of the id2 hetero - dimeric substrate . our findings provide key insights into the structural basis for the mono - ubiquitination of the id2 complex , the defining step in the activation of the fa / brca icl repair pathway . although the fancl subunit of the core complex is an active e3 ligase in isolation ( alpi et al . , 2008 ) , its mono - ubiquitination function is stimulated by 6-fold in the presence of fancb and faap100 ( rajendra et al . , 2014 ) . to understand whether the b100 association provides a structural framework for efficient mono - ubiquitination , or whether it causes a conformational change in fancl that stimulates its activity , we have expressed and purified human bl100 from insect cells . several steps of purification yielded a pure trimeric complex that eluted with a sharp peak under gel filtration and with an apparent molecular weight of 440 kda ( figures 1a and s1 ) . em imaging confirmed that our preparation is highly homogeneous and monodisperse ( figures 1b and 1c ) . two - dimensional classification of negative stain data revealed an elongated bow - tie structure containing apparent 2-fold symmetry ( figure 1d ) . the particle consists of a compact center and two globular modules , mapping at the two extremities of the assembly . cryo - em analysis yielded classes that were highly reminiscent of those obtained after negative stain imaging ; however , no significant improvement in resolution could be detected , hinting at an inherent flexible nature in the bl100 complex ( figure 1e ) . to test our hypothesis that bl100 is mobile and can adopt different structural states , we have performed focused classification on the central core of the structure , followed by classification on the whole particle without further alignment . this approach revealed that the globular domains at the two poles in the structure move as rigid bodies , either tilting or stretching , relative to the central module ( figure s1 ; movie s1 ) . despite this flexibility , the apparent 2-fold symmetry is maintained in the central core and at the two poles in the structure . from the data shown so far , it remains unclear whether bl100 is , indeed , a 2-fold symmetric assembly or , rather , contains a pseudo-2-fold symmetry axis , similar to id2 ( figure 1f ) . to address this issue , we have repeated the two - dimensional analysis , using a fancl derivative that contains maltose - binding protein ( mbp ) fused to the n terminus ( figures 2a and s2 ) . mbp tags provide an electron - dense feature that can be averaged in negative stain em , and they serve as pointers to localize individual proteins in a macromolecular assembly ( chen et al . , 2008 ) . we reasoned that a bona fide 2-fold symmetric complex should show two ( fancl - fused ) mbp densities that are dyad related . indeed , as evident from two - dimensional class averages , b-(mbp)l100 is decorated with two symmetry - related mbp densities , establishing that the three factors assemble into a homo - dimer ( b : l:100 = 2:2:2 stoichiometry , with an expected molecular weight of 468 kda , coherent with our analytical gel filtration profile ) ( figures 2b and 2c ) . a dimeric nature of the fa core complex was unexpected and invites a mechanistic model for the id2 mono - ubiquitination reaction that is further detailed in the discussion section . previous reports indicate that ring domains can dimerize ( brzovic et al . , 2001 , 2011 ) , and our mbp analysis shows that the ring - containing fancl subunit maps in proximity of the dimerization interface of bl100 ( figures 2b and 2c ) . to test whether the fa core complex dimerization requires fancl , we have expressed and purified a subunit dropout assembly that only contains fancb and faap100 for single - particle analysis ( figures 2d and s3 ) . in the absence of fancl , the dimerization interface remains intact , while the globular poles in the assembly become markedly flexible ( figures 2e and s3 ; movie s2 ) , revealing a structural role for fancl in stabilizing the bl100 assembly ( figure 2f ) , in line with previous reports ( alpi et al . , we have attempted to purify the isolated fancb subunit , but we found that this factor is insoluble when expressed in the absence of faap100 ( data not shown ) . conversely , the faap100 subunit is soluble and monodisperse in the absence of fancb , according to analytical gel filtration and single - particle em ( figures 2 g and s3 ) . comparison of faap100 with the b100 two - dimensional class averages , centered on the flexible globular pole component , allows us to recognize faap100 as the element mapping at the extremities of the b100 particle . no resemblance was detected between the isolated faap100 and the dimerization core in the assembly ( figures 2h and s3 ) . from these data , we conclude that fancb and faap100 provide a dimeric scaffold to position fancl on opposite poles in the protein assembly ( figure 2i ) . this suggests that efficient substrate ubiquitination requires a 2-fold symmetric arrangement in the fa core complex . furthermore , our subunit dropout experiments are incompatible with a homo - dimerization role for either fancl or faap100 , leaving the fancb subunit as the sole candidate for the dimerization element of the bl100 complex . analytical biochemistry combined with single - particle em analysis allowed us to propose a model for the molecular architecture of a dimeric bl100 assembly . difference mapping suggests that fancb is located at the homo - dimerization interface ; however , we failed in our attempts to directly show that the isolated fancb can self - associate . therefore , we turned to xl / ms analysis , a powerful tool to study the architecture of macromolecular assemblies , especially when combined with structural em ( bui et al . , 2013 ) ( figure 3a ; table s1 ) . we have used an amino - reactive bi - functional crosslinker ( bs3 ) to covalently bridge between surface - exposed primary amines that map within a distance range of 2630 between c of lysine residues . samples were crosslinked and trypsinized , enriched for larger crosslinked peptides through gel filtration , and analyzed by nanolc - ms ( nano - liquid chromatography - mass spectrometry ) using an ltq orbitrap velos . we first validated the xl / ms data by comparing our results with the available crystal structure of the human fancl central domain . all detected crosslinks in fancl exist within solvent - exposed lysines that are spaced within a range compatible with the dimensions of the crosslinker spacing arm ( figure 3b ) . this control provided us with confidence in the interaction network built for the rest of the protein assembly . remarkably , fancb contains , by far , the highest frequency of intra - molecular crosslinks , followed by fancl ( figures 3a and 3c ) . possible explanations for this deficiency include the unusually low number of lysines in this subunit ( 1.9% of the sequence , with 5.7% being the average in human proteins ) , their location in large peptides lacking trypsin - cleavage sites , or the absence of proximal lysines in the tertiary structure of faap100 . instead , the majority of crosslinked lysines observed for faap100 were involved in inter - molecular crosslinks with fancb . altogether , these data provide additional support to the notion that fancb maps at the homo - dimerization interface in the core complex ( figures 2 and 3c ) . we note that one of the intra - fancb crosslinks detected involves surface - exposed lysines ( in position 35 and 39 ) , which flank a known fa - associated mutation ( l37s ) . given the immediate proximity of lys35 and lys39 , leu37 is likely solvent exposed and could serve a role in interacting with other fa proteins . further confidence in our xl / ms results is derived from the striking match between the inter - molecular crosslinks and our biochemical / em analysis of the bl100 assembly . for example , four crosslinks connect faap100 with fancb , in accord with our finding that fancb - faap100 can exist as an isolated assembly ( figures 3a and 3c ) . fancl and fancb are connected by five crosslinks , which agrees with the observation that the mbp n - terminally fused to fancl maps in close proximity to the central fancb homo - dimer ( figure 2b ) . conversely , fancl and faap100 are not crosslinked , in agreement with our findings that ( 1 ) co - expression of fancl and faap100 does not yield a l100 hetero - dimer ( data not shown ) and that ( 2 ) the two subunits only appear loosely associated in the globular pole in the bl100 particle ( figure 2i ) . in conclusion , xl / ms analysis , combined with our single - particle em , provides a compelling architectural description of the dimeric bl100 catalytic assembly of the fa core complex . we have , so far , established that the bl100 catalytic assembly of the fa core complex forms a homo - dimer . whether this arrangement extends to the rest of the core complex , or whether the full assembly contains a symmetry mismatch element , remains to be established . therefore , we decided to purify the cef assembly and image it in isolation ( as an mbp - fancc fusion derivative to increase the overall molecular mass of the complex ) ( figures 4a and s4 ) . ( mbp)cef appears as an electron - dense , tri - lobed entity with no detectable 2-fold symmetry component ( figures 4b and s4 ) . we have then reconstituted a bl100-(mbp)cef co - assembly ( figures 4c and s4 ) and found that particles maintain the characteristic dimeric architecture of bl100 and are decorated with either one or two cef additional densities , connected to the globular poles in the bow - tie structure ( figures 4d and s4 ) . therefore , binding of two copies of cef to the bl100 complex is physically possible , indicating that the bl100 dimer acts as an organizing center for other core complex components . whether two copies of ag20 are found in the full fa core complex remains to be established and will be the focus of future studies . in the bl100-cef complex , cef projects outward from the bl100 dimerization center and appears perfectly poised to latch onto the outer perimeter of the id2 mono - ubiquitination substrate ( figure 4d ) . a direct contact between fance and fancd2 has been previously reported ( gordon et al . , 2005 , pace et al . , 2002 , 2014 ) , and in an accompanying study , we show that human cef can bind both fanci and fancd2 directly ( van twest et al . , 2017 ) . reconstituting a cef - id2 assembly could , therefore , provide key insights into the mechanism of the fanci - fancd2 mono - ubiquitination by the fa core complex . xenopus laevis id2 can be expressed and purified to homogeneity and efficiently processed in the mono - ubiquitination reaction by the human fa core complex ( van twest et al . to gain mechanistic insights into id2 mono - ubiquitination by the human fa core complex , we chose to produce xenopus id2 for single - particle em studies ( figure s5 ) . in agreement with recently published work ( liang et al . , 2016 ) , we observed that id2 forms an asymmetric , flexible , dimeric complex , which resists averaging and high - resolution structure determination ( figure s5 ) . we hypothesized that cef binding might stabilize the id2 assembly , and we co - expressed the five proteins in the attempt to purify an id2-cef complex by affinity purification via a flag - tagged fanci subunit . this purification strategy , including stringent ion exchange and size exclusion chromatography ( sec ) steps , yielded an apparently stoichiometric id2 assembly , according to coomassie - stained sds - page gel analysis , but only trace amounts of the cef assembly ( figure s6 ) . as expected from inspection of the sds - page gel , the vast majority of id2 particles show no sign of cef co - complex , and no clear evidence for cef association was detected by inspection of the two - dimensional classes . to our surprise , however , id2 particles from the cef co - expression experiments yielded neatly averaging two - dimensional classes ( figure s6 ) . a pseudo-2-fold symmetric end - on view can be recognized in the assembly and perfectly matches the two - dimensional projections of an electron density map generated from the pdb coordinates of the mouse id2 crystal structure ( pdb : 3s4w ) ( joo et al . , 2011 ) , we repeated the co - expression experiment using a different cef derivative , containing a strep ii tag on fancc ( figure 5a ) . single - particle analysis on the reconstituted complex confirms our initial observations ( figures 5b5d and s6 ) . to obtain a three - dimensional structure of the stabilized id2 assembly , we generated an initial model using eman2 , followed by three - dimensional classification , and auto - refined the most populated three - dimensional class to 18.3 resolution using relion ( figure 6b ; figure s7 ) . rigid - body docking of the mouse atomic structure into the xenopus id2 map results in an excellent fit , with a striking match at the hetero - dimerization interface ( figure 6b ) . conversely , minor adjustments would be needed in the c - terminal region of the two protomers to perfectly fit the atomic structure into the em density ( although we chose not to perform these local adjustments , given the limited resolution of our three - dimensional em data ) . coherent with a more flexible nature of the fanci and fancd2 carboxy - terminal modules , analysis of complex dynamics based on two - dimensional class averages of end - on views indicates that the peripheral lobes in the id2 dimer are flexible ( movie s3 ) . in summary , our characterization of the id2-cef interaction reveals that the two protein assemblies do not form a stoichiometric complex , at least in the experimental conditions tested . remarkably , however , cef co - expression alters the configuration of the id2 dimer , stabilizing the dimerization interface and locking the assembly in a pseudo-2-fold symmetric structure ( figure 5d ) . this unexpected observation has key implications for the mechanism of id2 mono - ubiquitination and suggests a role for the cef assembly of the fa core complex . because the two strands of dna are antiparallel ( watson and crick , 1953 ) , the factors that spatially organize , cut , or open the double helix are often arranged as symmetric dimers . for example , a dyad element can be found in the protein core of the nucleosome ( the building block of chromatin in eukaryotic cells ) ( richmond et al . , 1984 ) , in dna integrases ( which concertedly cut the two dna strands to insert foreign dna ) ( engelman and cherepanov , 2014 ) , or in dna helicases that unwind the double helix ( li et al . , 2015 ) . this 2-fold symmetric architecture can then be propagated to a subsequent set of proteins that physically interact with the dna - binding dimer . a paradigm for this concept is found in the eukaryotic origin of dna replication , where two replisomes are built symmetrically around a dimer of ring - shaped helicases ( araki , 2016 , costa et al . , 2014 , deegan et al . , 2016 ) by a set of regulatory and chaperone proteins that are , in turn , symmetric dimers ( itou et al . , 2015 ) . in our study , we report one additional example of how a 2-fold symmetric enzyme modifies a dimeric dna interactor . in this case , a homo - dimeric regulator protein acts symmetrically on a hetero - dimeric assembly . in the fa / brca icl repair pathway , the fanci - fancd2 dimer binds the double helix at the damaged site and acts as a landing platform that recruits a set of dna - repair proteins for icl processing ( ceccaldi et al . , 2016 ) . fanci and fancd2 are structural homologs that can arrange in a pseudo-2-fold - symmetric assembly ( joo et al . , 2011 ) . recruitment of the dna - repair machinery requires mono - ubiquitination of both factors ( smogorzewska et al . , 2007 ) . this function is provided by the fa core complex , an enzyme assembly whose architecture was previously unknown ( ceccaldi et al . , 2016 , walden and deans , 2014 ) . here , we showed that the trimeric center of the fa core complex ( bl100 ) exists as a symmetric homo - dimer ( figure 2c ) . fancb and faap100 play an architectural role , orienting the two e3 ligases in an ideal position to symmetrically target the fanci and fancd2 ubiquitination sites ( figure 2i ) . in particular we note that fancb is the only protomer in the bl100 catalytic module that contains disease - associated missense mutations , emphasizing the key role of this factor in the assembly of a functional fa core complex ( chandrasekharappa et al . , 2013 ) inspection of em two - dimensional class averages reveals that the bl100 particle is dynamic , with the two catalytic poles of the complex free to tilt and stretch with respect to the fixed fancb dimerization center ( figure s1 ; movie s1 ) . remarkably , this movement occurs symmetrically across the particle ( when the upper lobe moves to the left , the lower lobe moves to the right , and vice versa ) . if the catalytic subunit fancl is omitted , however , the two external lobes in the particle move independently of each other ( figure s3 ; movie s2 ) . although the functional significance of this concerted movement is unclear , it is evident that each of the two catalytic subunits in the assembly must be able to sense the state of the symmetry mate in the assembly , and this allosteric communication must be mediated by the fancb dimerization center . in summary , our study provides the first insights into the mechanics of this dimeric e3 ligase super - assembly and starts to explain how the fancl mono - ubiquitination activity can be stimulated by the fancb and faap100 architectural factors ( rajendra et al . , 2014 ) . the bl100 assembly also functions as the organizing center of the fa core complex , providing a dimerization scaffold for the recruitment of a set of two cef trimeric assemblies ( figures 4d and s4 ) . the fance subunit of cef has been previously reported to interact with the id2 assembly ( gordon et al . , 2005 , pace et al . , 2002 , polito et al . , 2014 ) , and our structural data suggest that cef might act as a molecular matchmaker to bridge between the id2 mono - ubiquitination substrate and the bl100 e3 ligase machinery . previous structural studies suggest that the id2 dimer might exist in different configurations during the ubiquitination reaction . for example , crystallization of the murine complex captured a symmetric , compact state of the id2 assembly , with the ubiquitination sites mapping at the dimerization interface , probably impairing access to the ubiquitination machinery ( joo et al . , 2011 ) . conversely , a cryo - em study of the human id2 shows that the complex is more flexible ( poorly averaged ) in solution ( liang et al . , 2016 ) . in agreement with these recent findings , we show that the intact xenopus id2 complex expressed in isolation can not be captured in discrete conformational states ( two - dimensional images do not average ) ( figure s5 ) . in contrast , the co - expression of cef with id2 appears to stabilize a symmetric state of id2 , which is highly reminiscent of the form captured in the crystal structure ( figures 5c and 6 ; movie s3 ) . this hints at a possible role for cef in manipulating the id2 substrate to facilitate symmetric mono - ubiquitination of the two related protomers ( figure 7 ) . biochemical analysis of the reconstituted id2 mono - ubiquitination reaction by the fa core complex supports our suggestion that the cef assembly acts as a nexus between the bl100 enzyme and the id2 substrate while also reconfiguring the id2 structure ( van twest et al . , 2017 ) . whereas the isolated bl100 assembly solely ubiquitinates the fancd2 subunit , the presence of cef significantly stimulates fanci mono - ubiquitination , necessary for triggering initiation of the fa / brca icl repair pathway . how bl100-cef and id2 interact and influence one another remains to be explained at a molecular level . we note that a disease - associated mutation on fance ( r371w ) , predicted to destabilize the fance fold , abrogates the interaction with fancd2 in a yeast two - hybrid study . the same arg - to - trp point mutation on fancd2 ( r302w ) , mapping within the outer perimeter of the n - terminal id2 dimerization module , likewise abrogates the fance - d2 interaction ( joo et al . , 2011 , such elements likely play an important role in the cef - mediated stabilization of the id2 substrate during the mono - ubiquitination reaction catalyzed by the homo - dimeric bl100 catalytic module . notably , in addition to interacting with fance , the same r302 element in fancd2 has been previously implicated in mediating a histone h3 contact ( sato et al . , how the id2 mono - ubiquitination reaction occurs in the context of chromatinized dna is an exciting frontier topic . several outstanding questions will need to be addressed to understand the mechanism of activation of the fa / brca icl repair pathway . for example : how do disease mutations affect the structure and function of the fa core complex ? what is the molecular mechanism for the formation of the fa core complex in cells ? our discovery that the fa core complex is a symmetric dimer changes the way we think about these problems . baculoviruses were constructed as detailed in the supplemental information . for the expression of the fancb - fancl - faap100 complex and the isolated faap100 , 1 l of sf9 cells at a titer of 2.0 10 cells per milliliter were suspended in 4 l spinner flasks ( corning ) and infected with baculovirus at an moi of 2 for 72 hr at 27c . cells were collected post - infection by centrifugation ( 5,000 rpm , 4c , 15 min ) , washed in pbs buffer supplemented with 5 mm mgcl2 , and resuspended in lysis buffer ( 25 mm hepes naoh [ ph 7.6 ] , 15 mm nacl , 0.02% tween 20 , 10% glycerol , 1 mm edta , 1 mm egta , 2 mm mgcl2 , 2 mm 2-bme , 0.4 mm pmsf , and roche protease inhibitor cocktail ) . the suspension was lysed by dounce homogenization ( 40 strokes , tight pestle ) , and nacl was added to a final concentration of 250 mm . the lysate was cleared by centrifugation ( 15000 rpm , 4c , 35 min ) , and the supernatant was incubated ( 3 hr , 4c ) with anti - flag m2 affinity resin ( sigma aldrich ) pre - equilibrated with flag washing buffer ( 25 mm hepes naoh [ ph 8.0 ] , 200 mm nacl , 0.02% tween 20 , 10% glycerol , 1 mm edta , 1 mm egta , and roche protease inhibitor cocktail ) . after wash , the sample was eluted with flag elution buffer ( 25 mm hepes naoh [ ph 8.0 ] , 100 mm nacl , 0.02% tween 20 , 5% glycerol , 1 mm edta , 1 mm egta , 0.3 mg / ml flag peptide , and roche protease inhibitor cocktail ) . elution fractions were combined and loaded onto a mono q 5/50 gl for ion - exchange chromatography ( ge healthcare ) . the column was washed with q - wash buffer ( 25 mm hepes naoh [ ph 8.0 ] , 150 mm nacl , 5% glycerol , 1 mm edta , 1 mm egta , 0.5 mm tcep , and roche protease inhibitor cocktail ) and eluted over a 150-mm to 650-mm nacl linear gradient in the same buffer . the peak fraction from cation - exchange chromatography was loaded onto a superose 6 pc 3.2/30 column ( ge healthcare ) operating on an ktamicro hplc ( high - performance liquid chromatography ) system ( ge healthcare ) and was equilibrated in 25 mm hepes naoh ( ph 8.0 ) , 250 mm nacl , and 1 mm tcep . the proteins were separated by sds - page , using 4%12% bis - tris gels ( invitrogen ) running for 50 min at 150 v in mops buffer or using 3%8% tris - acetate gels ( invitrogen ) running for 60 min at 150 v in tris - acetate buffer . protein concentration was determined by measuring absorbance at 280 nm ( a280 nm ) using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies ) . expression of the fancb - faap100 and fancb-(mbp)fancl - faap100 complexes were performed using the fancb - fancl - faap100 protocol ; however , infection was performed using an moi of 1 for 72 hr at 27c . freshly purified samples ( bl100 at 1 mg / ml , b100 at 0.25 mg / ml , and faap100 at 1.5 mg / ml ) were immediately used for negative - stain em or cryo - em experiments . 1 l of sf9 cells at a titer of 1.0 10 cells per milliliter was suspended in 4-l spinner flasks ( corning ) and infected with baculovirus at an moi of 1 for 72 hr at 27c . cells were collected post - infection by centrifugation ( 5,000 rpm , 4c , 15 min ) , washed in pbs buffer supplemented with 5 mm mgcl2 , and resuspended in lysis buffer ( 25 mm hepes naoh [ ph 7.6 ] , 15 mm nacl , 0.02% tween 20 , 10% glycerol , 1 mm edta , 1 mm egta , 2 mm mgcl2 , 15 mm 2-bme , 0.6 mm pmsf , and roche protease inhibitor cocktail ) . the suspension was lysed by dounce homogenization ( 40 strokes , tight pestle ) , and nacl was added to a final concentration of 200 mm . the lysate was cleared by centrifugation ( 15,000 rpm , 4c , 35 min ) , and the supernatant was incubated ( 2 hr , 4c ) with amylose resin ( new england biolabs ) pre - equilibrated with amylose wash buffer ( 25 mm hepes naoh [ ph 8.0 ] , 150 mm nacl , 0.02% tween 20 , 10% glycerol , 1 mm edta , 1 mm egta , 0.25 mm tcep , and roche protease inhibitor cocktail ) . after extensive wash , the sample was eluted by ligand competition using the amylose wash buffer supplemented with 20 mm maltose ( sigma ) . peak fractions were pooled and loaded onto a mono q 5/50 gl for ion - exchange chromatography ( ge healthcare ) . the column was washed with buffer q - a ( 50 mm hepes naoh [ ph 8.0 ] , 150 mm nacl , 5% glycerol , 1 mm edta , and 0.5 mm tcep ) and eluted over a linear salt gradient with buffer q - b ( 50 mm hepes naoh [ ph 8.0 ] , 1 m nacl , 5% glycerol , 1 mm edta , and 0.5 mm tcep ) . peak fraction was successively loaded onto a superose 6 pc 3.2/30 column ( ge healthcare ) operating on an ktamicro hplc system ( ge healthcare ) and separated by sec in 25 mm hepes naoh ( ph 8.0 ) , 100 mm nacl , and 2 mm dtt . sample concentration was measured by a280 nm using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies ) and the ( mbp)cef theoretical extinction coefficient . freshly purified ( mbp)cef sample at a final concentration of 0.5 mg / ml was immediately used for preparation of negative - stain em grids or reconstitution with freshly purified bl100 sample . briefly , 1 l of sf9 cells at a titer of 2.0 10 cells per milliliter was suspended in 4-l spinner flasks ( corning ) and co - infected with the two baculoviruses , each at moi of 1 , for 72 hr at 27c . following the co - infection , cells were collected by centrifugation ( 5,000 rpm , 4c , 15 min ) and washed in pbs buffer supplemented with 5 mm mgcl2 . cells were resuspended in lysis buffer ( 25 mm hepes naoh [ ph 7.6 ] , 15 mm nacl , 0.02% tween 20 , 10% glycerol , 1 mm edta , 1 mm egta , 2 mm mgcl2 , 2 mm 2-bme , 0.4 mm pmsf , and roche protease inhibitor cocktail ) and lysed by dounce homogenization ( 40 strokes , tight pestle ) . to preserve complex integrity , the lysate was added with nacl to a final concentration of 150 mm and cleared by centrifugation ( 15,000 rpm , 4c , 35 min ) . the supernatant was collected and incubated ( 3 hr , 4c ) with anti - flag m2 affinity resin ( sigma - aldrich ) pre - equilibrated in flag washing buffer ( 25 mm hepes naoh [ ph 8.0 ] , 120 mm nacl , 0.02% tween 20 , 10% glycerol , 1 mm edta , 1 mm egta , and roche protease inhibitor cocktail ) . after extensive wash , the proteins were eluted by incubation of the resin in flag elution buffer ( 25 mm hepes naoh [ ph 8.0 ] , 120 mm nacl , 1 mm edta , 0.3 mg / ml flag peptide , and roche protease inhibitor cocktail ) . peak fractions were combined and loaded onto a mono q 5/50 gl(ge healthcare ) column running in buffer q - a ( 25 mm hepes naoh [ ph 8.0 ] , 100 mm nacl , 5% glycerol , 1 mm edta , and 0.25 mm tcep ) . elution of the sample was performed over a linear salt gradient with buffer q - b ( 25 mm hepes naoh ph 8.0 ] , 750 mm nacl , 5% glycerol , 1 mm edta , and 0.5 mm tcep ) . to increase sample homogeneity , the complex was further purified by pull - down of the strep - tag ii on fancd2 . peak fraction from ion - exchange chromatography was incubated for 2 hr at 4c with strep - tactin superflow plus resin ( qiagen ) . the resin was washed in strep - wash buffer ( 25 mm hepes naoh [ ph 8.0 ] , 120 mm nacl , 10% glycerol , 1 mm edta and 0.25 mm tcep ) and eluted by incubation with strep - wash buffer supplemented with 25 mm desthiobiotin . fractions containing stoichiometric i - d2 complex were pooled and concentrated on amicon ultra mwco 10k centrifugal filters ( millipore ) . concentrated sample was separated by size exclusion on a superdex 200 pc 3.2/30 column ( ge healthcare ) operating on an ktamicro hplc system ( ge healthcare ) running in 25 mm hepes naoh ( ph 8.0 ) , 120 mm nacl , and 1 mm tcep . elution peak was analyzed by sds - page followed by instant blue safestain staining ( expedeon ) . freshly purified id2 sample at a final concentration of 0.25 mg / ml was immediately used for preparation of negative - stain em grids . co - expression of fanci - fancd2-fancc - fance - fancf was obtained by infecting sf9 cells with baculoviruses encoding for fanci , fancd2 , and either mbp - tagged or strepii - tagged cef . briefly , 1 l sf9 cells at a titer of 2.0 10 cells per milliliter were co - infected with baculoviruses encoding for flag - fanci , strepii - fancd2 , and ( mbp)cef ( or ( strepii)cef ) , each at a moi of 1 . protein extraction , purification , and identification were carried out as previously described for the id2 complex . the id2-(strepii)cef sample purified from ion - exchange chromatography and the id2-(mbp)cef sample purified from sec were used for preparation of em grids by negative stain immediately after purification . the reconstitution of the fancb - fancl - faap100-(mbp)fancc - fance - fancf complex was performed by mixing equal volumes of a freshly purified 1-m solution of bl100 and a 3-m solution of cef ( resulting in a molar ratio of 1:3 ) and loaded on a 30k mwco slide - a - lyzer dialysis cassette ( thermo scientific ) . the protein mix was first dialyzed for 1 hr at 4c in buffer 500 ( 25 mm tris - hcl [ ph 8.0 ] , 500 mm nacl , 5% glycerol , and 1 mm dtt ) , then for 2 hr in buffer 350 ( 25 mm tris - hcl [ ph 8.0 ] , 350 mm nacl , 5% glycerol , and 1 mm dtt ) , and finally overnight in buffer 100 ( 25 mm tris - hcl [ ph 8.0 ] , 100 mm nacl , 5% glycerol , and 1 mm dtt ) . the sample was recovered from the dialysis cassette and loaded onto a superose 6 pc 3.2/30 column ( ge healthcare ) operating on a ktamicro fplc ( fast protein liquid chromatography ) system ( ge healthcare ) equilibrated in 25 mm tris - hcl ( ph 8.0 ) , 100 mm nacl , and 2 mm dtt . in control experiments , isolated dialyzed bl100 and ( mbp)cef complexes were also subject to sec as described for the reconstituted samples . samples were analyzed by sds - page on 3%8% criterion xt tris - acetate gels ( bio - rad ) followed by instant blue safestain staining ( expedeon ) . a 2- to 6-nm - thick carbon layer was evaporated onto freshly cleaved mica sheets ( ems ) using a q150te carbon coater ( quorum technologies ) . carbon was incubated overnight at 50c before floating on 600-mesh copper grids ( agar scientific ) or quantifoil r1.2/1.3 300-mesh copper grids . prior to sample incubation , the carbon - coated grid was glow discharged for 30 s at 45 ma using a 100 glow discharger ( ems ) . serial dilutions of each protein sample were performed to achieve optimal particle concentration on the em grid ( final concentrations ranged from 30 to 5 g / ml ) . a 4-l drop of the freshly purified sample solution was applied onto the glow - discharged grid and incubated for 1 min . approximately 2 l of sample solution was absorbed by gentle side blotting , and the grid was immediately stained with four subsequent applications onto 4 50-l drops of a 2% ( wt / vol ) uranyl formate solution with gentle stirring for 8 s. after staining , the grid was blotted dry and stored at room temperature prior to imaging . particles of reconstituted bl100-(mbp)cef , isolated ( mbp)cef , isolated id2 , and id2 co - expressed with ( mbp)cef were imaged on a tecnai g2 spirit lab6 transmission electron microscope ( tem ; fei ) operating at 120 kev ( electron microscopy science technology platform , the francis crick institute ) . images were manually acquired on a ultrascan 1000 2k 2k charge - coupled device ( ccd ) camera ( gatan ) at a nominal magnification of 30,000 ( corresponding to a pixel size of 3.45 at the specimen level ) . particles of bl100 , b100 , b-(mbp)l100 , isolated faap100 , and id2 co - expressed with ( strepii)cef samples were imaged using a jem-2100 lab6 electron microscope ( jeol ) operated at 120 kv . images were recorded on a ultrascan 4k 4k ccd camera ( gatan ) at a nominal magnification of 42,000 corresponding to a pixel size of 2.73 at the specimen level . on both microscopes , images were collected in low - dose mode ( electron dose of 35 e / ) and with defocus values in the range of 0.5 m to 2.0 m . for the bl100 and faap100 samples , the micrographs were automatically recorded using the jeol automated data acquisition system ( jadas ) with same electron dose and defocus value as the manual collection . a total of 567 images were collected for the bl100 sample , 250 images for the b100 sample , 412 images for the b-(mbp)l100 sample , 540 images for the faap100 sample , 277 images for the ( mbp)cef sample , 455 images for the bl100-(mbp)cef sample,712 images for the id2 sample , 468 images for the id2-(mbp)cef sample , and 447 images for the id2-(strepii)cef sample . ultrathin carbon - coated lacey 300-mesh copper grids ( ted pella ) were glow discharged for 30 s at 45 ma using a 100 glow discharger ( ems ) and mounted onto a vitrobot mark iv ( fei ) . a 4-l drop of freshly purified bl100 at a concentration of 20 the sample was incubated on the grid for 1 min in 100% humidity at 21c . after incubation , the grid was double - side blotted for 2.0 s and rapidly plunged in liquid ethane . cryo - em data for the bl100 sample were collected on a titan krios tem ( fei ) operated at 300 kev and equipped with a back - thinned falcon ii ( fei ) direct electron detector at a nominal magnification of 47,000 , resulting in a pixel size of 1.77 at the specimen level . a total of 2,798 images were recorded at defocus values in the range of 2.54.5 m and with a cumulative electron dose of 56 e / distributed over 26 frames . beam - induced motion was corrected on each image using the motioncorr program ( li et al . , 2013 ) . micrographs showing contamination drift , or out - of - range defocus values were discarded . for each dataset , particles were semi - automatically picked using the eman2 package , version 2.12 ( tang et al . , 2007 ) . from the negative - stain em data collection , a total of 63,543 individual particles were extracted from the bl100 dataset , 63,365 particles from the b100 dataset , 51,941 particles from the b-(mbp)l100 dataset , 177,106 particles from the faap100 dataset , 36,768 particles from bl100-(mbp)cef dataset , 47,898 particles from the ( mbp)cef dataset , 95,553 particles from the id2 dataset , 27,084 particles from the id2-(mbp)cef dataset , and 71,295 particles from the id2-(strepii)cef dataset . from the bl100 cryo - em dataset , the parameters of the contrast transfer function ( ctf ) were estimated on each micrograph using ctffind3 ( mindell and grigorieff , 2003 ) for negative - stain data and ctffind4 ( rohou and grigorieff , 2015 ) for cryo - em data . reference - free two - dimensional alignment and averaging was performed using relion , version 1.4 ( scheres , 2012 ) . for three - dimensional reconstruction of the id2 complex co - expressed with ( strepii)cef , an initial three - dimensional volume was generated using the e2inimodel.py program in the eman2 package ( tang et al . , 2007 ) . the initial volume was used as a reference model for three - dimensional classification and refinement in relion 1.4 ( scheres , 2012 ) . the atomic model of the fanci - fancd2 hetero - dimer ( pdb : 3s4w ) ( joo et al . , 2011 ) was used for rigid - body fitting into the em map using ucsf ( university of california , san francisco ) chimera ( pettersen et al . , 2004 ) . the refined em map of the id2 hetero - dimer has been deposited in the three - dimensional - em database ( http://www.emdatabank.org ) with accession number : emd-3476 . all chemicals were purchased from sigma at the highest grade possible , unless otherwise stated . nano - lc - ms solvents and additives were purchased as lc - ms grade from fisher scientific . the xl / ms workflow was conducted as previously described ( leitner et al . , 2014 ) . briefly , bl100 dimer , at a concentration of 0.5 mg / ml in 25 mm hepes ( ph 8) , 300 mm nacl , 10% glycerol , and 0.25 mm tris(2-carboxyethyl)phosphine ( tcep ) , was reacted with 5 mm bs3 ( using a 50:50 ratio of d0 and d4 ) for 30 min at 37c . the reaction was quenched by adding 50 mm ammonium bicarbonate for 20 min at 37c and then dried to completion using vacuum centrifugation . the sample was resuspended at a concentration of 1 mg / ml using 8 m urea , reduced with 2.5 mm tcep , and alkylated with 5 mm iodoacetamide . the sample was diluted with 50 mm ammonium bicarbonate to reduce the urea concentration to 1 m , and trypsin ( promega ) was added at a 1:50 enzyme : substrate ratio and left to digest overnight at 37c . the solution was then acidified , and peptides were purified using c18 in house solid - phase elution tips ( empore , 3 m ) . for size exclusion fractionation , the peptides were resuspended in sec buffer ( 70% h2o , 30% acetonitrile , and 0.1% trifluoroacetic acid ) , and chromatographically separated using an ktamicro ( ge healthcare ) with a superdex peptide pc 3.2/30 column ( ge healthcare ) and detected using 215 nm absorbance . the flow rate was set to 50 l / min , and 100 l fractions were collected and dried to completion using vacuum centrifugation . for nano - lc - ms acquisition , the sec fractions were resuspended into 30 l of 0.1% trifluoroacetic acid in h2o , and 1 l was injected and chromatographically resolved using an ultimate 3000 rslcnano ( dionex ) with an easy - spray column ( 2-m particles , pepmap c18 , 100- pore size , 50 cm 75 m i d ) ( thermo scientific ) . a flow rate of 0.25 l / min was used , starting at 98% mobile a ( 0.1% formic acid , 5% dmso , 95% h2o ) and 2% mobile b ( 0.1% formic acid , 5% dmso , 80% acetonitrile , 10% h2o ) . over 80 min , mobile b was then increased to 40% followed by a further increase to 90% over 10 min . spectra were acquired in real time using an ltq orbitrap velos mass spectrometer ( thermo scientific ) with a 400 to 2,000 m / z ( mass - to - charge ratio ) acquisition window . the top ten most intense ions with a charge state of + 3 or greater were then selected for tandem ms ( ms / ms ) using data - dependent acquisition and cid ( collision - induced dissociation ) fragmentation ( at 35% normalized collision energy ) . a dynamic exclusion list , with a repeat count of 1 , repeat duration of 20 s , exclusion list size of 100 , and exclusion duration of 30 s , was used to prevent repeat sampling . the data were searched for bs3 mono - links ( + 156.079 da for water quench , + 155.096 da for ammonium quench ) , intra - links , and intra- and inter - protein crosslinks ( + 138.068 da ) on lysine residues , using isotopic pairing with a mass shift of + 4.025 da for bs3 d4 ions , in addition to a fixed carbamidomethylation modification ( + 57.021 da ) and variable methionine oxidation ( + 15.994 da ) . the data were searched using a 10-ppm mass tolerance for charge states + 3 or above . xprophet was then used , and peptides with a false discovery rate ( fdr ) below 5% were then manually assessed for adequate signal intensity and peak assignment . , s.v.t . , and f.s . constructed all baculoviruses and performed preliminary protein preparations . optimized purification protocols for structural studies , prepared all proteins and em grids , collected em data ( with l.r . ) , and performed all image analysis under the supervision of a.c .

summaryactivation of the main dna interstrand crosslink repair pathway in higher eukaryotes requires mono - ubiquitination of fanci and fancd2 by fancl , the e3 ligase subunit of the fanconi anemia core complex . fanci and fancd2 form a stable complex ; however , the molecular basis of their ubiquitination is ill defined . fancd2 mono - ubiquitination by fancl is stimulated by the presence of the fancb and faap100 core complex components , through an unknown mechanism . how fanci mono - ubiquitination is achieved remains unclear . here , we use structural electron microscopy , combined with crosslink - coupled mass spectrometry , to find that fancb , fancl , and faap100 form a dimer of trimers , containing two fancl molecules that are ideally poised to target both fanci and fancd2 for mono - ubiquitination . the fancc - fance - fancf subunits bridge between fancb - fancl - faap100 and the fanci - fancd2 substrate . a transient interaction with fancc - fance - fancf alters the fanci - fancd2 configuration , stabilizing the dimerization interface . our data provide a model to explain how equivalent mono - ubiquitination of fanci and fancd2 occurs .

Introduction Results Discussion Experimental Procedures Author Contributions

PMC2957246

compared to normal - weight adults , overweight and obese persons have not only greater bone mineral density ( bmd ) , but they lose bone at a slower pace , and may even have a reduced risk of fragility fractures [ 1 , 2 ] . as regards the effect of childhood obesity on bone mass , findings are inconsistent [ 35 ] . some studies have attributed greater bone mass to excess body weight in the growing years [ 58 ] but it has also been suggested that obese children have lower bone mass for a given weight [ 911 ] . in principle , the greater bone mass in obese subjects could simply be a consequence of increased body mass . while the weight load per se can stimulate bone formation , obese subjects also have moderate increase in muscle mass [ 12 , 13 ] , which is an important determinant of bone mass and strength . in absolute terms bone mass and strength have been shown to be greater in obese women but reduced relative to body weight varying in proportion to lean mass , not to body weight or fat mass . interestingly , some studies have suggested that fat mass , the other major component of body weight , may also stimulate bone accrual in growing children , but these results have remained inconsistent showing both positive [ 16 , 17 ] and negative associations [ 11 , 13 , 18 ] . despite the apparent simplicity , while obesity is represented by increased body weight , and thus the mass needed to move during habitual activities , the incident muscular contractions , particularly the magnitude of force , the rate of force production , and the total amount of contractions play a more important role than body weight . body weight alone imposes relatively small and static load on bones ( corresponding to earth 's gravity , g ) , whereas the load can be substantially amplified in different activities by muscle actions . theoretically , a physically active obese individual has stronger muscles than a sedentary , similarly obese person . consequently , the greater muscle force would permit more bone remodeling and thus result in increased bone mass and strength in the former . on the other hand , if an obese individual is sedentary or becomes inactive and loses muscle mass , incident muscle forces imposed on bones decline and osteopenia could ensue no matter how obese the individual might be . however , it is not yet known whether the influence of increased body weight on bones would be stronger in childhood and persist until adulthood . the conventional paradigm suggests that obesity results in greater bone mass as a consequence of greater body weight and could thus protect against osteoporosis . in this cross - sectional study we hypothesized that those women who have been obese since childhood would show some benefit in bone traits compared to women who have gained extra weight not earlier than in adulthood . this hypothesis was based on observations that the period of adolescent growth provides the window of opportunity for efficient bone accrual . in addition , we evaluated whether statistical adjusting for different anthropometric variables affects the results , and if so , to what extent . sixty - two obese ( bmi > 30 ) clinically healthy premenopausal women with the age range of 25 to 45 years participated in the study . according to self - reports , 12 of these women had been obese since childhood , and 50 had gained excess weight after maturity . none of the subjects had evidence for any disease , prior injuries , or drug use ( hormonal contraceptives were allowed ) that would have affected their skeleton . the study protocol was approved by the ethics committee of the pirkanmaa hospital district , tampere , finland , and each participant gave her written informed consent . information on self - reported health , injuries , medication , diseases , diet , nonpregnant weight at the age of 25 , history of weight cycling , weight loss attempts , menstrual status , and lifestyle factors such as current physical activity , smoking , and consumption of alcohol was obtained with a questionnaire . physical activityhistory of physical activity was assessed in an interview , and current daily walking steps were measured with a pedometer ( omron hl-112-e , omron healthcare europe ) on six days . history of physical activity was assessed in an interview , and current daily walking steps were measured with a pedometer ( omron hl-112-e , omron healthcare europe ) on six days . muscle performancethe maximal isometric leg extension force ( n / kg ) was measured by a strain gauge dynamometer at a knee angle of 110 degrees ( tamtron , tampere , finland ) . the maximal take - off force ( n / kg ) and power ( w / kg ) during a vertical counter - movement jump were measured on a force - plate ( kistler ergojump 1.04 , kistler instrumente ag , winterthur , switzerland ) . functional agility ( s ) was evaluated by a figure-8 running test and the grip strength of both forearms ( kg ) with a standard grip strength meter . the maximal isometric leg extension force ( n / kg ) was measured by a strain gauge dynamometer at a knee angle of 110 degrees ( tamtron , tampere , finland ) . the maximal take - off force ( n / kg ) and power ( w / kg ) during a vertical counter - movement jump were measured on a force - plate ( kistler ergojump 1.04 , kistler instrumente ag , winterthur , switzerland ) . functional agility ( s ) was evaluated by a figure-8 running test and the grip strength of both forearms ( kg ) with a standard grip strength meter . anthropometry and body compositionbody height was measured to the nearest 0.1 cm and body weight to the nearest 0.1 kg with a high - precision scale with the participants wearing only their underwear . waist circumference was measured midway between the lowest rib and the iliac crest and hip circumference at the tip of the greater trochanter . body composition ( fat mass and lean mass ) and the android ( trunk ) and gynoid ( hip ) fat-% were assessed with dual - energy x - ray absorptiometry ( dxa , lunar prodigy advance , ge lunar , madison , wi , usa ) . in our laboratory , the in vivo precision ( coefficient variation , cv% ) based on repeated scans of 27 subjects with repositioning is 1.3% for the fat mass and 0.8% for the lean mass ( sievnen , unpublished data ) . body height was measured to the nearest 0.1 cm and body weight to the nearest 0.1 kg with a high - precision scale with the participants wearing only their underwear . waist circumference was measured midway between the lowest rib and the iliac crest and hip circumference at the tip of the greater trochanter . body composition ( fat mass and lean mass ) and the android ( trunk ) and gynoid ( hip ) fat-% were assessed with dual - energy x - ray absorptiometry ( dxa , lunar prodigy advance , ge lunar , madison , wi , usa ) . in our laboratory , the in vivo precision ( coefficient variation , cv% ) based on repeated scans of 27 subjects with repositioning is 1.3% for the fat mass and 0.8% for the lean mass ( sievnen , unpublished data ) . bone mass and structurebone mineral content ( bmc , g ) of the total body , bmc , and bone mineral density ( bmd , g / cm ) of the left proximal femur and lumbar spine were assessed with dxa ( lunar prodigy advance ) . femoral neck structure was assessed using the advanced hip analysis ( aha ) which provided data on cross - sectional area occupied by bone mineral ( csa , mm , an index of bone strength against compression ) , section modulus ( z , mm , an index of bone strength against bending ) , and outer diameter ( width , mm ) . in our laboratory , the in vivo precision is 1.4% for the total body bmc , about 1% for the lumbar spine bmc , 1.5% for the femoral neck bmc , and about 3% for the trochanter bmc ( sievnen 2005 , unpublished data ) . the in vivo precision for csa , z , and width is 2.3% , 3.8% , and 1.2% , respectively.in addition to the dxa measurements , the left radius and tibia were measured with pqct ( norland / stratec xct 3000 , pforzheim , germany ) . the tomographic slices were taken from the shaft and distal part of the tibia ( 50% and 5% from the distal endplate of the tibia , resp . ) and of the radius ( 30% and 4% from the distal endplate of the radius , resp . ) according to our standard procedures . for the shaft regions , the analyzed bone traits were total cross - sectional area ( toa , mm ) , cortical density ( cod , mg / cm ) , and density - weighted polar section modulus ( ssi , mm , an index of bone strength against bending ) . for the distal parts of the radius and tibia the traits were toa , trabecular density ( trd , mg / cm ) , and ssi . in our laboratory , the in vivo precision of the pqct traits is between 0.7% ( cod of the tibial shaft ) and 7.7% ( ssi of the distal radius ) . bone mineral content ( bmc , g ) of the total body , bmc , and bone mineral density ( bmd , g / cm ) of the left proximal femur and lumbar spine were assessed with dxa ( lunar prodigy advance ) . femoral neck structure was assessed using the advanced hip analysis ( aha ) which provided data on cross - sectional area occupied by bone mineral ( csa , mm , an index of bone strength against compression ) , section modulus ( z , mm , an index of bone strength against bending ) , and outer diameter ( width , mm ) . in our laboratory , the in vivo precision is 1.4% for the total body bmc , about 1% for the lumbar spine bmc , 1.5% for the femoral neck bmc , and about 3% for the trochanter bmc ( sievnen 2005 , unpublished data ) . the in vivo precision for csa , z , and width is 2.3% , 3.8% , and 1.2% , respectively . in addition to the dxa measurements , the left radius and tibia were measured with pqct ( norland / stratec xct 3000 , pforzheim , germany ) . the tomographic slices were taken from the shaft and distal part of the tibia ( 50% and 5% from the distal endplate of the tibia , resp . ) and of the radius ( 30% and 4% from the distal endplate of the radius , resp . ) according to our standard procedures . for the shaft regions , the analyzed bone traits were total cross - sectional area ( toa , mm ) , cortical density ( cod , mg / cm ) , and density - weighted polar section modulus ( ssi , mm , an index of bone strength against bending ) . for the distal parts of the radius and tibia the traits were toa , trabecular density ( trd , mg / cm ) , and ssi . in our laboratory , the in vivo precision of the pqct traits is between 0.7% ( cod of the tibial shaft ) and 7.7% ( ssi of the distal radius ) . between - group differences were evaluated by an analysis of covariance , and as possible confounding factors body height , fat , and lean mass were used as covariates . since the body mass index ( bmi ) is a commonly used covariate in the pertinent literature , the analyses were adjusted for bmi for comparison . linear regression analyses were used to find the predictors for bone traits ; body height , lean mass , fat mass , and childhood obesity were included as the dichotomous variable ( obc = 0 ; oba = 1 ) in the regression models . between - group differences were evaluated by an analysis of covariance , and as possible confounding factors body height , fat , and lean mass were used as covariates . since the body mass index ( bmi ) is a commonly used covariate in the pertinent literature , the analyses were adjusted for bmi for comparison . linear regression analyses were used to find the predictors for bone traits ; body height , lean mass , fat mass , and childhood obesity were included as the dichotomous variable ( obc = 0 ; oba = 1 ) in the regression models . the obc - group was on average 5.2 cm taller and compared to the oba - group they had 2.5 kg and 3.5 kg more lean and fat mass , respectively . however , difference in mean relative body fat and lean mass was small indicating similar body composition in both groups for given body height in adulthood . as shown in table 1 , the muscle strength , functional agility , and steps / day were similar between the two groups . the dxa - based bone mass and strength were greater in the obc - group than that those in the oba - group ( table 2 ) . adjusting for bmi had practically no effect on the mean differences , whereas the adjustment for height , fat , and lean mass affected the results , which were no more statistically significant . although the obc - group tended to have greater trd both at the distal radius and at the distal tibia , cortical density was similar in both groups . there was also a tendency for greater total bone cross - sectional area in obc - group especially in the weight - bearing tibia . however , this difference disappeared after adjusting for body height and fat and lean tissue . in the nonweight - bearing radius , cross - sectional bone area was similar in both groups , and adjusting for anthropometric variables did not affect the results . the most significant predictors for bone traits were height and lean mass ( tables 3 , 4 , and 5 ) . height was positively associated with bone mass and strength at the axial skeleton , while lean mass was the strongest correlate for most bone traits at the appendicular skeleton . according to our findings , women who had been obese since childhood were taller than women who had gained weight in adulthood . obese children tend to be tall for their age and advanced in sexual and skeletal development , but not necessarily in terms of adult height , which was found in the present study . since body size is strongly associated with bone mass and strength , and taller people have more massive skeleton than their smaller counterparts , body height and weight , or their aggregate measure bmi , are commonly used in literature as covariates to statistically control for the between - group differences in anthropometric variables . besides the conventional variables , we measured both fat and lean tissue ( the main components of body weight ) in this study , and both of these variables were used as covariates instead of pure body weight . according to our findings , body height is a much stronger predictor of bone mass and strength than body weight , while lean mass turned out to be a stronger predictor for pqct - assessed appendicular bone traits than height . taller persons usually have bigger bone cross - sections , and in line with this notion the total csa of the weight - bearing tibia was greater in the obc group than in the oba group . in this respect , it is recalled that bmi ( by definition = weight /height in meters ) does not totally correct differences in body size , which was also shown by our results . thus we argue that bmi is not an appropriate variable in adjusting for different body sizes . in general , we found that bmi - adjusted dxa- and pqct traits were similar to unadjusted crude differences . however , after adjusting for body height , lean , and fat mass the mean differences declined and were no statistically significant . with regard to bone traits which are basically size - independent , such as cortical density , the adjustment did not affect the results . to sum up , if it is not possible to separate lean , and fat mass from each other , use of total body weight , in addition to height , better takes into account the differences in body size than bmi , and the former approach is recommended . increased biomechanical loading of the skeleton due to increased body weight and increased lean mass ( ~muscle forces ) may contribute to greater bone mass and size . obese persons ( both children and adults ) have to move greater body mass in habitual physical activity compared to the persons with normal body weight , which implies greater loading on bones provided that the intensity and the amount of exercise are similar . some previous dxa - based studies , but not all , have shown that obese children have normal or increased bone mass relative to healthy weight peers [ 4 , 6 ] . in contrast , goulding et al . found decreased bone mass in obese children relative to bone size and body weight . also a canadian cohort showed that the greater bone strength in obese children was appropriate for their lean mass , not their fat mass . in a recently published pqct - study the obese children had similar cortical density but greater radial and trabecular density than normal weight children , which gives support to our finding . some previous studies have also suggested that fat mass may modulate periosteal bone growth in childhood [ 16 , 23 ] , but we did not find such associations in adulthood , the weak correlation with the bone strength index at the distal radius excluded . due to cross - sectional study design , we can not definitely say whether the obc and oba groups differed essentially in their habits of physical activity in childhood ( commuting to school , playing , and related activities ) . however , at the time of the study the participants were 40 years old on average , which means that when they were school kids in 1970s , a decade when children in finland commonly walked or cycled to school , played outdoor games , spent less time watching tv , and had no internet games or computers . this being the apparent case , their bones received versatile dynamic loading in childhood and thus an adequate mechanical stimulus . in contrast , it is well possible that when the present - day children are grown - up , the situation is different . nowadays children are more and more transported to school by car or bus , and instead of playing outdoor games , they are engaged in computer games or internet in their leisure activities . the obesity epidemic is associated with sedentary life style , which means substantially less dynamic loading on bones and most likely more fragile bone in adulthood . similarly we can only speculate about hormonal effects on bone development in childhood and adolescence . however , extra weight in the form of fat mass , besides its potential mechanical influence on bones [ 12 , 14 ] , may increase production of many hormones , such as estrogen , leptin , and insulin [ 13 , 24 ] . adipose tissue is known to express aromatase enzymes that convert steroid precursors to estrogen , which has been reported to stimulate and suppress periosteal bone growth in childhood . obese children may enter puberty earlier than normal - weight children , and they also achieve similar estradiol levels and bone ages at significantly earlier chronological ages than nonobese children [ 26 , 27 ] . hormonal actions may thereby potentially contribute to the increased linear growth and skeletal mass observed in childhood obesity . further studies are , however , needed to elucidate effects of adipose tissue and adipose - modulated hormones on adult bone mass and strength . the strength of this study was the use of pqct besides the conventional dxa , which allowed not only the differentiation of trabecular and cortical bone but also information on bone geometry . in addition , bone traits both at the weight - bearing tibia and nonweight - bearing radius were evaluated , and the muscle performance of the subjects was assessed . obviously this rendered the study underpowered to statistically uncover meaningful differences , such as the properly adjusted 5 to 8% differences in trabecular density . second , our study group represented a convenience sample and focused only on premenopausal women . third , the analyses were based on a self - reported retrospective data , and we do not have exact body weight data from childhood over the adolescent period . fourth , the obc group was about 5 cm taller , which may be just a coincidence . childhood obesity has been shown to be associated with greater height - for - age , advanced maturation for age , and greater lean mass for height , although the advantage in growth gradually decreases and probably does not account for taller height in adulthood . it is , however , recalled that the difference in body height was statistically controlled for and it did not affect the present conclusions . in conclusion , childhood obesity seemed to be slightly associated with denser trabecular bone in adulthood in both weight - bearing and nonweight - bearing bones among obese premenopausal women , but not with cortical density , neither was childhood obesity associated with total cross - sectional bone area . it is recalled that the study sample was small and the between - group mean differences did not reach statistical significance . nevertheless , the results bring up an interesting question , whether childhood obesity can result in denser trabecular bone in adulthood . finally , in this kind of research attention must be paid on proper adjustment of the data .

associations between childhood obesity and adult bone traits were assessed among 62 obese premenopausal women , of which 12 had been obese since childhood ( obc ) , and 50 had gained excess weight in adulthood ( oba ) . body composition and bone mineral content ( bmc ) of the total body , spine , and proximal femur were assessed with dxa . total cross - sectional area and cortical ( diaphyseal cod ) and trabecular ( epiphyseal trd ) bone density of the radius and tibia were measured with pqct . compared to oba - group , obc - group was 5.2 cm taller having 2.5 and 3.5 kg more lean and fat mass , respectively . depending on the statistical adjustment , obc - group had 510% greater trd both in tibia and in radius . the remaining bone traits did not significantly differ between the groups . current preliminary observations bring up an interesting question whether childhood obesity can result in denser trabecular bone in adulthood . however , prudence must be exercised in the statistical adjustment .

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

PMC5165164

significantly increased mortality rates and decreased life quality are observed in patients who have experienced fractures . owing to their high socioeconomic relevance and increasing incidence , apart from the estimated costs of around 5.4 billion euros a year , therapeutic alternatives need to be investigated . sarcopenia , a loss of skeletal muscle mass , emerges during the development of osteoporosis [ 36 ] . this results in a higher risk of falls and , consequently , fall - related injuries [ 7 , 8 ] . ninety percent of all fractures in elderly people arise from falls . a variety of endocrine substances synthesised by muscle which influence the bone are reduced in sarcopenia . for instance , levels of muscle - derived humoral bone anabolic factors , which activate osteoblasts , are decreased . this implies that a sarcopenic muscle is an accompaniment of osteoporosis and the occurrence of osteoporotic fractures . histologically , sarcopenia leads to a reduction in size of both muscle fibre types i and ii , whereas the fast type ii fibres are chiefly affected [ 1214 ] . former studies suggested that a low grade inflammation contributes to sarcopenia ; however , the detailed underlying mechanisms remain unclear . the endocrine axis between muscle and bone , consisting of insulin - like growth factor 1 , interleukins ( ils ) 6 , 7 , and 15 , and osteoglycin , and vice versa for sclerostin with its muscle counterparts myostatin and osteocalcin , is impaired in osteoporosis and , therefore , in sarcopenia they seem to be two sides of the same coin [ 1619 ] . recent studies also showed a significant loss of number , thickness , and capillarization of muscle fibres with a majority of type ii fibre loss [ 2022 ] . therefore , therapeutic possibilities reside in pharmacological intervention which target impaired regenerative capacity , elevated reactive oxygen species production , and inflammation observed in sarcopenic muscle . several of these inflammation reactions are mediated by lipoxygenases , enzymes of arachidonic acid metabolism . baicalein , as a phytochemical agent , is extracted from the plant scutellaria baicalensis georgi . it acts as a lipoxygenase ( especially cyclooxygenase ( cox - i ) inhibitor ) and is also a potent inhibitor to 12-lipoxygenase ( 12-lox ) and 15-lox and , therefore , has an antioxidative effect [ 2427 ] . furthermore , it inhibits nuclear factor kappa - light - chain - enhancer of activated b cells ( nf-b ) and suppresses the function of tumor necrosis factor - alpha 8 ( tnf- ) and/or il-6 , all of them being mediators of inflammation cascades . an effect of baicalein on inflammatory degenerative bone diseases , such as rheumatoid arthritis , through its suppression of apoptosis and secretion of matrix metalloproteinases , has been recently described [ 28 , 29 ] . baicalein , as a common flavonoid , also inhibits eotaxin production , probably having a positive effect on asthma . additionally , 12-lox is inhibited by baicalein , where the former increases the activity of hif-1 under hypoxic conditions , causing the upregulation of the vascular endothelial growth factor , a major factor in promoting prostate cancer progression and metastasis . proposal . as the effect of baicalein on sarcopenia has not been clarified in previous studies , we aimed to decipher whether baicalein could reduce sarcopenic symptoms in muscles of osteoporotic rats . outcome measures included the number and diameter of muscle fibres , the relation with type i , type iia , and type iib muscle fibres , and the number of capillaries in muscles ( the relation of capillaries to muscle fibre ) . furthermore , serum levels of calcium , magnesium , and creatine kinase , a possible biomarker for sarcopenia , were measured to estimate the general muscle condition . for the study , 61 female , 3-month - old sprague dawley rats ( winkelmann company ) were kept in cages at 20c and at a relative humidity of 55% in makrolon iv. after 1 week of acclimatization , the experiments were conducted in accordance with the ethical standards of animal care and approved by the local district government ( application number : g14/1530 ) . at 13 weeks of age , the rats underwent bilateral ovariectomy ( ovx ) or were left intact , as previously described . surgical procedures were carried out under ketamine / domitor anaesthesia ( 0.1 ml/100 g body weight ( bw ) , intraperitoneal ( i.p . ) ) . in brief , after shaving , anaesthesia , and disinfection , the skin was incised on both sides of the lower abdomen . after 8 weeks , osteotomy of tibia metaphysis with plate osteosynthesis was performed for different studies , according to . if the standardized tibia osteotomy , performed bilaterally in all rats , would have any effect on muscles , it would be similar in all treatment groups . on the basis of previous studies [ 35 , 36 ] , we assumed that , at around this time point , that is , 8 weeks after ovx , the rats would have developed osteoporosis with sarcopenia . baicalein ( 98% , sigma - aldrich chemie gmbh , munich ) was dissolved in 100% dimethyl sulfoxide ( dmso ) , while both control groups ( non - ovx and ovx , each n = 10 ) received dmso alone . baicalein was injected subcutaneously at different concentrations as previously described [ 37 , 38 ] ( c1 : 1 mg / kg bw , c2 : 10 mg / kg bw , and c3 : 100 mg / kg bw , resp . ) in three groups with 10 animals each . m. gastrocnemius , m. soleus , and m. longissimus were removed , and m. gastrocnemius and m. soleus were weighed . afterwards , m. gastrocnemius and m. soleus were sectioned transversely in half across the whole muscle to make cryotome - cutting easier . subsequently , 12 m thick frozen sections were prepared by cutting the muscle samples orthogonally with a cryotome ( cm 1900 ; leica microsystems ) . to identify the distribution and changes in types i and ii fibres , atpase staining was carried out on muscle sections , as described by hork . moreover , periodic acid - schiff ( pas ) staining to visualize capillaries in muscle and subsequent counting was performed according to the method described by andersen et al . for capillary counting , two sections of each muscle in pas staining were chosen and , in a 0.25 mm square , every capillary and muscle fibre were counted , which taken together delivered the ratio of capillary / muscle fibre . for fibre measurements , in three different image sections of each muscle in atpase staining , 30 type i , 30 type iia , and 30 type iib fibres were edged and area and diameter were determined by the program nis - elements ar 4.0 ( nikon instruments europe , amsterdam , netherlands ) . after that , in 1 mm fibre , types were counted and their relation calculated . after the rats were sacrificed , blood samples of 0.5 ml were collected and analysed with the creatine kinase assay using a c16000 analyser ( abbott , wiesbaden , germany ) for quantitative measurement of serum values . calcium was measured with the same analyser at 660 nm using arsenazo iii dye and magnesium at 572 nm after the calcium was complexed and eliminated with a chelating agent so as to not interfere with the measurements . statistical analyses were conducted using graphpad prism ( version 5.04 , graphpad software , inc . , san diego , ca ) . for detecting differences between the different groups , one - way anova ( f test , differences between individual means were estimated using tukey 's test ( = 0.05 ) . in all figures , the mean values and the standard error of the mean ( sem ) are displayed . to test the effects of ovariectomy and different concentrations of baicalein on body weight , the rats were weighed on the day of receipt ( figure 2(a ) ) and at postmortem ( figure 2(b ) ) . there was no significant difference between groups in body weight on the day of receipt . however , a significant difference in postmortem weight was noted between the non - ovx and ovx groups . the postmortem weights of m. gastrocnemius ( figure 2(d ) ) and m. soleus ( figure 2(e ) ) were not statistically different ; the weight of m. longissimus was not detected . to test the effects of ovariectomy on the uterus , the uteri of non - ovx rats weighed approximately 0.6 g and were significantly heavier than those collected from the other groups ( on average , 0.10.2 g ) . these findings indicate successful ovariectomy with resulting atrophy of the uterus in all ovx groups . in a previous study , since sarcopenia is associated with lower skeletal muscle capillarization , the effect of baicalein on muscle capillarization was interesting ; however , the effect of baicalein on muscle capillarization has not been described yet . we analysed the number of capillaries per 0.5 mm muscle tissue in m. soleus ( 3a ) , m. gastrocnemius ( figure 3(b ) ) , and m. longissimus ( figure 3(c ) ) . although we could not see differences between non - ovx and ovx animals , rats treated with baicalein showed significantly higher numbers of capillaries per muscle fibre when compared with the untreated groups . on average , the rate of capillarization moreover , the group treated with the highest concentration of baicalein ( c3 ) showed significantly more capillaries per fibre when compared with the group treated with the lowest concentration ( c1 ) . after atpase staining , muscle fibres were analysed for their diameter and area . in total , 90 fibres of each type for each sample were assessed , and 90% of all cells were type i ; therefore , no further differentiation was performed . to assess characteristics of sarcopenia , we determined the diameters and areas of the muscle fibres . for m. soleus , although the diameter in non - ovx rats was on average 63 m , it was reduced in the ovx cohort ( approximately 57 m ; figure 4(a ) ) . compared with ovx group , baicalein c2- and c3-treated groups possessed single fibres with significantly increased diameters ( 6568 m ) . muscle fibre areas were calculated after preparing muscle sections ( figure 4(b ) ) ; 90 fibres per muscle were assessed . the ovx group showed a decrease in area ( 2,511 m ) when compared with the non - ovx control group ( 3,161 m ) . upon comparing the baicalein - treated groups to ovx groups , significantly greater areas were found in the c2 and c3 groups ( 3,408 m and 3,630 m , resp . ) . effects of baicalein on area and diameter were confirmed when observing the results in relation to body weight ( figures 4(c ) and 4(d ) ) . assessment of single muscle weight in relation to muscle diameter showed no significant differences ( data not shown ) . when relating muscle fibre diameter and area to muscle weight , no significant differences , but similar tendencies to the relation to body weight , were seen . this is perhaps because of relatively low muscle weight , which could lead to measurement inaccuracy ( figures 4(b ) and 4(e ) ) . in m. gastrocnemius , type i fibres showed an increased diameter of approximately 20% when treated with baicalein compared with the non - ovx group ( figure 5(a ) ) . a similar effect could be observed in type iia fibres , with the strongest effect in the c2 group ( figure 5(b ) ) . by contrast , type iib fibres showed no significant differences among all groups ( data not shown ) . regarding the size of the area of different fibre types , in type i ( figure 5(c ) ) , compared with the non - ovx control group , all ovx cohorts ( treated and untreated ) showed an increase in fibre area . compared with the non - ovx group , ovx rats showed an increase in area of 18% , and baicalein - treated animals showed an increase of up to 41% . c2 cohort showed the highest effect , but only c2 and c3 were statistically significant . a similar tendency was observed in type iia fibres ; however , only c2 group possessed a significantly greater area ( figure 5(d ) ) . no significant differences were observed in the area of single type iib muscle fibres ( data not shown ) . thereafter , we calculated the diameter to body weight ratio and found no significant differences in types i ( figure 5(e ) ) , iia , and iib fibres ( data not shown ) . similar results were obtained when relating diameter to muscle weight for types i ( figure 5(f ) ) , iia , and iib fibres ( data not shown ) , where no significant differences could be determined . moreover , we calculated the relation of fibre area to body weight . in type iia fibres , we found a slight increase in m. gastrocnemius area in group c2 when compared with ovx rats ; however , other groups did not show significant differences ( figure 5(g ) ) . overall findings for types i and iib fibres indicated no striking differences between the groups ( data not shown ) . when relating area to muscle weight , we found significant differences in type i fibres , where non - ovx , ovx , and c1 groups did not show significant differences ( figure 5(h ) ) . however , when comparing their values to those of the c3 group , baicalein c3 treatment could increase the area by approximately 60% . similar effects were observed for type iia fibres , where the highest baicalein concentration increased the area by approximately 44% ( figure 5(i ) ) . in iib fibres , a similar tendency was observed ; however , the differences were not significant ( figure 5(j ) ) . comparable with m. gastrocnemius , the three fibre types could also be differentiated for m. longissimus . when observing the diameter in m. longissimus type i fibres , we did not detect significant differences between non - ovx and ovx rats ; however , high doses of baicalein ( c2 ) could raise the diameter by approximately 13% ( figure 6(a ) ) . when comparing the effects of different baicalein concentrations , the strongest effects were recorded in the c2 group . similar effects were observed in type iia fibres , where baicalein c2 could increase the diameter to 86.7 m when compared with 77.6 m in non - ovx and 74.8 m in ovx cohorts ( figure 6(b ) ) . the iib fibre analysis showed no significant differences among the different groups ( data not shown ) . we measured the area of muscle fibres types i and iia and , analogous to the diameter , similar values were obtained for non - ovx and ovx animals ( figures 6(c ) and 6(d ) ) . by contrast , baicalein treatment could significantly increase the muscle fibre area . while all baicalein concentrations increased the area compared with the control animals , c2 rats showed the highest values . type iib fibres showed no significant differences among the cohorts ( data not shown ) . unlike the analysis of m. soleus and gastrocnemius , we related the fibre diameter and area in m. longissimus to animal body weight because single muscle weight was not detected . type i fibre diameter was significantly decreased after ovariectomy ( figure 6(e ) ) ; however , after baicalein treatment ( c2 ) , this effect could be rescued . the same phenomenon was observed for types iia ( figure 6(f ) ) and iib ( figure 6(g ) ) fibres . when relating the areas of types i and iia fibres to body weight , a similar decrease could be observed in ovx rats when compared with the non - ovx control group ( figures 6(h ) and 6(i ) ) . when treating the animals with low ( c1 ) or high ( c3 ) baicalein concentrations , the effect of the ovariectomy was dampened ; however , fibre areas were lower than those in the non - ovx cohort . in contrast , medium baicalein concentrations ( c2 ) could not only rescue the consequences of the ovariectomy but also increase the fibre areas when compared with the non - ovx rats . for iib fibres , no significant differences between the groups could be measured ( data not shown ) . finally , the proportions of different fibre types ( type i , iia , and iib ) were assessed in m. longissimus ( figure 7 ) . in general , no striking differences were observed among the different groups . the strongest effects were noted in animals treated with the medium concentration of baicalein ( c2 ) . notably , when comparing the non - ovx and c2 cohorts , we detected that c2 rats tended to have less type i and type iib fibres but contained significantly more type iia fibres . in m. soleus and m. gastrocnemius , creatine kinase ( u / l ) , calcium ( ca ; mmol / l ) , and magnesium ( mg ; mmol / l ) levels were measured in the groups . creatine kinase levels were significantly lower in the c3 group than in the ovx group ( figure 8(a ) ) . a higher serum magnesium value could be detected in the c2 group when compared with the ovx group ( figure 8(c ) ) . sprague dawley rats are a valuable model for the induction of osteoporosis after hormonal depletion via ovariectomy [ 43 , 44 ] . here , the rats were 13 weeks old at the time of ovariectomy , which is reportedly acceptable for the osteoporosis model [ 45 , 46 ] . furthermore , francisco et al . compared the effects of ovariectomy in rats of different ages and found no related differences in bone mineral response . the effects on muscle in rats seem to be similar to those in humans : they develop sarcopenia [ 48 , 49 ] . to determine the effects of baicalein on sarcopenia independent of baicalein treatment , body weight of the rats after ovariectomy was significantly greater for the ovx ( control ) group than for the non - ovx group . interestingly , in m. gastrocnemius , an increasing dose of baicalein led to slightly reduced muscle weight , supporting the hypothesis of estrogen deficiency - induced muscle degeneration . however , the reduced muscle weight of m. gastrocnemius could also be attributed to the baicalein - induced inhibition of the nonphysiological degeneration of muscle , a finding reported in literature describing the anti - inflammatory and antifibrotic effects of baicalein . as reported previously , estrogen has an angioproliferative effect on skeletal muscle [ 51 , 52 ] and a loss of capillaries by 25% could be observed in sarcopenia because of aging [ 21 , 53 ] . notably , we could show that baicalein leads to a significant gain in the capillary to muscle fibre ratio in all baicalein - treated groups . in m. soleus , baicalein treatment showed a dose - dependent increase in the number of capillaries per muscle fibre . similar results could also be seen in m. gastrocnemius and in m. longissimus , where the 10 mg / kg bw concentration evinced the greatest effect on capillarization . the expected loss of capillaries under estrogen deficiency could not be observed when treated with baicalein . the hypothesis that baicalein , applied subcutaneously , stimulates angiogenesis in skeletal muscle despite estrogen deficiency was proven . nonetheless , there were no significant differences in capillary to muscle fibre ratio after ovx , which might be due to insufficient trial duration , which was too short to reveal the decapillarization in sarcopenic muscle , as seen in other rat models . we purported that decreased muscle diameter due to estrogen loss , as described in rats [ 5356 ] and humans , could be restored by baicalein . indeed , the baicalein c2 and c3 groups had significantly increased diameters of m. soleus , a finding similar to that for body weight . these findings support our hypothesis that baicalein counteracts muscle atrophy at high concentrations . in all three fibre types ( i , iia , and iib ) in m. gastrocnemius we saw a tendency of all baicalein - treated groups to have increased fibre diameters . interestingly , the group treated with baicalein c2 concentration showed the greatest fibre diameters . when analysed in detail , the diameter to body weight ratio showed that types i , iia , and iib fibres in almost all baicalein groups were slightly increased . we also found a tendency for greater diameter per body weight values in the non - ovx group when compared with the ovx group . overall , baicalein appears to induce hypertrophy in the estrogen - deficient skeletal muscle . in m. longissimus , the development of sarcopenia after ovariectomy , resulting in estrogen deficiency , which leads to reduced muscle fibre diameter is supported . altogether , the expected atrophy of skeletal muscle fibres , indicated by the decline in fibre diameter , appears to be rescued by baicalein . the gain of iia fibre diameter is noteworthy , considering that the diameters are typically affected in sarcopenia . the fact that lipoxygenase inhibitors could counteract muscle atrophy has already been shown in clinical trials . in m. longissimus , the increase in types i and iia fibre areas suggests that baicalein can counteract the estrogen - dependent atrophy . in m. gastrocnemius , the increase of type nonetheless , this gain in area and diameter can also be attributed to the storage of fat and water . moreover , estrogen deficiency can lead to an increase in noncontractile muscle fibres that can hardly be differentiated from normal muscle tissue . whether this is microscopically , no greater fat deposits or lipid content could be observed . in affecting muscle fibre types i , iia , and iib , baicalein appears to react with oxidative- and glycolytic - working muscle groups . in m. longissimus , significantly increased proportions of fast iia but not type i fibres were observed in the baicalein c2 group . this finding disagrees with the decline in fast fibres , the described fast - to - slow fibre type shift , which is characteristic for muscle atrophy and wasting . in conclusion , these results suggest a positive , though not fibre type - specific , effect of baicalein on estrogen - deficient muscles in rats . whereas in m. gastrocnemius and m. soleus the number of capillaries in muscle was proportional to the dose of baicalein , in m. longissimus , the medium baicalein concentration ( 10 mg / kg bw ) showed the greatest effect . the results of muscle diameter and area also showed a positive baicalein effect in comparison with the control groups . moreover , regarding fibre ratios , a gain in iia fibres was seen in m. longissimus , which contradicts the fast - to - slow fibre type shift . as observed in human studies , there was no difference between sarcopenic and control groups regarding calcium and magnesium levels [ 62 , 63 ] . serum concentration of creatine kinase , a clinical marker of muscle state [ 32 , 64 ] , in baicalein c3 group was found to be significantly decreased . investigating molecular mechanisms of baicalein treatment was not within the scope of this descriptive study , but bhattacharya et al . showed that skeletal muscle atrophy ( during aging and neurodegenerative diseases ) can be diminished with inhibition of 12-lox and 15-lox , while the activation of this pathway led to increased nadph oxidase activity , protein ubiquitination , and proteolytic degradation . they also demonstrated that baicalein reduces denervation - induced muscle atrophy in wild type but not in 12-lox and 15-lox knockout mice . therefore , baicalein chiefly operates through this pathway and is not arbitrated through the reduction of inflammatory response . possibly the main effect of baicalein in this study could be observed because of the above - mentioned 12-lox and 15-lox pathway inhibiting nadph oxidase activity and protein ubiquitination as well as another study by wang et al . demonstrates the multiple signaling pathways in which baicalein is involved ( e.g. , akt / mtor , erk 1/2 , nf-b , and calcineurin ) . the aim of this study was to elucidate whether baicalein , in different doses , applied subcutaneously , could neutralize the estrogen - induced loss of muscle and atrophy in the skeletal muscle of rats . we found that baicalein has a positive effect on estrogen - dependent atrophy of skeletal muscle . this inflammatory effect has not been reported so far but did not affect overall health , as was seen by the analyses of body weight , food intake , and general conditions ( clean coat , absence of porphyrin around eyes , etc . ) . the inflammatory processes were not part of this study and we could not confirm an anti - inflammatory effect after s.c . therefore , it is advisable to use an alternative route of administration , such as orally [ 50 , 68 ] or intravenously . to emphasize differences between baicalein and ovx groups , trial duration could be elongated and more groups could provide better conditions to find the ideal dose and route of baicalein administration . moreover , a healthy non - ovx group should be included . as we only investigated skeletal muscle , we can not exclude systemic effects . no fever or severe infections occurred throughout the experiments . however , the development of the necrotic lesions at the injection sites should be taken into consideration . smooth muscles and cardiomyocytes as well as infection parameters , such as crp , il-6 , and procalcitonin , could be further examined to rule out effects on these tissues . to exclude the fact that gains in area and diameter are attributed to fat and water retention , biochemical analyses of muscle fibres additionally , there could be a possible effect of osteotomy on muscle tissue , so these experiments could be repeated without this operative procedure ; however , this effect would exist in all rats . different models of muscle inflammation could also be used to further investigate the anti - inflammatory effect of baicalein . in addition , the bone of rats and its biomechanical and histomorphometric composition could also be evaluated , as recently done by li et al . , but were not part of this study . detection of the molecular mechanism underlying the effects of baicalein in rats was not part of this descriptive study . this discrepancy could be explained by baicalein - induced inhibition of the nonphysiological degeneration of muscle due to the anti - inflammatory , antifibrotic [ 50 , 72 ] , and antioxidant [ 66 , 73 ] effects of baicalein but should be further examined .

sarcopenia , a loss of muscle mass accompanying osteoporosis , leads to falls and fall - related injuries . baicalein , as a phytochemical agent , has an antioxidative and anti - inflammatory effect in muscle . in this study , sixty - one female sprague dawley rats were divided into five groups : four groups were ovariectomized ( ovx ) and one control group was nonovariectomized ( non - ovx ) . eight weeks after ovariectomy , three disparate concentrations ( 1 mg / kg body weight ( bw ) , 10 mg / kg bw , and 100 mg / kg bw ) of baicalein were applied subcutaneously daily in three ovx groups . mm . soleus , gastrocnemius , and longissimus were extracted ; their diameter , area , relation to body , and muscle weights as well as number of capillaries per fibre were recorded . in mm . soleus and gastrocnemius , the baicalein effect ( increasing number of capillaries per fibre ) was proportional to the dose applied . the fibre diameters and area under baicalein treatment were significantly greater compared to ovx and non - ovx groups . in m. longissimus , we observed a shift to type iia fibres . serum creatine kinase levels were significantly lower in highest baicalein concentration group . we conclude that baicalein can stimulate angiogenesis , though not fibre type - specific , in skeletal muscle and reduce the estrogen - related loss of fibre diameter and area in the skeletal muscle in rats . therefore , a protective effect of baicalein on muscle cells can be assumed .

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

PMC3325502

cognitive impairment is a core deficit of a number of neuropsychiatric disorders ( goldberg & green , 2002 ; weickert et al . 2000 ) and drugs that improve facets of cognition such as attention , learning , memory and executive functions are known as pces ( morein - zamir et al . these drugs alter neurotransmitter modulation of cognition leading to improvements in cognitive deficits in patients with traumatic brain injury ( tbi ) ( teitelman , 2001 ) , depression ( vaishnavi et al . 2006 ) , addiction ( shearer & rodgers , 2009 ) , multiple sclerosis ( kraft & bowen , 2005 ; zifko et al . 2002 ) , parkinson 's disease ( nieves & lang , 2002 ) , and those suffering from alzheimer 's disease ( ad ) , schizophrenia , and attention deficit hyperactivity disorder ( adhd ) . there is substantial opportunity for meeting the challenge of improving cognition and mental wellbeing in those with mental health problems and for reducing substantially the factors that contribute to the loss of mental , social and economic capital ( beddington et al . a good illustration is ad , which is a neurodegenerative disorder characterized by a decline in cognitive and behavioural functioning . it is the commonest cause of dementia and one of the most disabling and burdensome health conditions worldwide ( ferri et al . there are currently 820 000 people with dementia in the uk , which costs 23 billion per year ( alzheimer 's society , 2007 ) . globally , if no effective prevention strategies or neuroprotective medications are developed , 81.1 million people will suffer from dementia by 2040 ( ferri et al . however , a treatment that would reduce severe cognitive impairment in older people by just 1% a year would cancel out all estimated increases in the long - term care costs due to ageing population ( comas - herrera et al . cognitive enhancers hold significant benefits in ameliorating these cognitive impairments in ad and countering these economic and social burdens ( alzheimer 's research trust , 2010 ; kaduszkiewicz et al . for example , cholinesterase inhibitors ( chei ) , such as donepezil , that inhibit centrally active acetylcholinesterase ( ache ) and boost acetylcholine in the brain , compensate for the degeneration of neurons in the neocortex that regulate attention and memory ( stahl , 2009 ) , and are effective in the treatment of mild and moderate ad ( eagger et al . interfere with amyloid deposition , or modulate nicotinic receptors ( pepeu & giovannini , 2009 ; stahl , 2009 ) while future drugs may exert their beneficial effects by activating various neurotransmitters including noradrenaline ( na ) , dopamine ( da ) , serotonin ( 5-ht ) , gaba and glutamate ( keowkase et al . furthermore , drugs that activate synaptic nmda receptors work synergistically with ampa receptors to produce long - lasting changes in the synaptic functioning and enable the encoding of new memories . this promotes the phosphorylation of creb that slows down the pathological changes observed in ad ( snyder et al . 2005 ) . however , these drugs only minimize the neural damage caused by glutamate 's neurotoxic effects and evidence is needed of improved episodic memory in ad patients . importantly , some drugs have significant side - effects . for example , donepezil is contra - indicated for people with liver problems ( mount & downton , 2006 ) while others have modest clinical efficacy in early ( lanctot et al . furthermore , about 3040% of patients with ad may not respond to chei , and approximately 29% of patients treated with chei leave clinical trials because of adverse events ( birks , 2006 ) . therefore , it is important to develop novel and effective neuroprotective agents that selectively target the underlying neuropathology associated with amnestic mild cognitive impairment and ad . developing these drugs could be advantageous to the individual and to society , particularly given the significant ageing population in the uk and the usa . it is not within the scope of this review to cover in detail the range and action of all the current and novel pces and therefore the reader is referred to stahl ( 2009 ) and the academy of medical sciences ( 2008 ) . notwithstanding , pces might also improve the quality of life in patients with tbi , which is the most common cause of disability in young people ( colantonio et al . for example , survivors of tbi often suffer from chronic cognitive deficits ( salmond et al . 2005 , 2006 ) in areas such as sustained attention and learning which implicate impaired cholinergic function ( polo et al . furthermore , voxel - based morphometry studies reveal structurally reduced grey - matter density and changes in hippocampus and neocortex in tbi patients . this is further consistent with the cholinergic dysfunction account that commonly contributes to the development of tbi - induced cognitive impairments ( salmond et al . consequently , the use of cheis that increase cholinergic function may be of benefit to tbi patients ( tenovuo et al . . however , whether pces improve apathy , which is often considerably disabling and detrimental to rehabilitative efforts in tbi patients , needs to be determined in future research ( keenan et al . equally , patients with schizophrenia can also benefit from pces through improvements in executive functions ( barnett et al . schizophrenia is a complex , lifelong disorder that significantly impairs cognitive and motivational function in approximately 1% of the world 's population . although psychotic symptoms , such as hallucinations and delusions , can be managed with antipsychotic treatment ( canuso et al . 2009 ) , patients continue to suffer dysfunctions in cognition , affect and motivation , which account for substantial decrements in social and occupational functioning ( harvey et al . 2007 ; matza et al . specifically , evidence indicates that these patients are substantially impaired in a wide range of neuropsychological task performances ( heinrichs & zakzanis , 1998 ; reichenberg & harvey , 2007 ) , and these impairments often impede everyday function and quality of life for many patients ( morein - zamir et al . pces may prove beneficial as an add - on to antipsychotic medication , as it has been suggested that , in patients with schizophrenia , even small improvements in cognitive functions , such as enhancing the ability to adapt efficiently to new situations and to plan effectively , could help them make the transition to independent living ( altamura & glick , 2010 ; davison & keefe , 1995 ) . enhancing patients cognition will not only improve their quality of life , but will also enable them to access jobs and integrate with society . in consequence , governments are relieved from the cost burden of ongoing care for these patients ( nicholl et al . in keeping with this , we have shown that pce drugs improve cognitive performance in patients with schizophrenia ( barnett et al . 2010 ) , and that modafinil ( provigil ) , a wake - promoting drug licensed for narcolepsy , can improve cognitive flexibility as measured by extra - dimensional attentional set - shifting in patients with chronic schizophrenia ( turner et al . 2004a ) . recently , modafinil has been shown to enhance some aspects of social cognition such as emotional facial recognition in patients with first episode of psychosis ( scoriels et al . however , many of the studies cited above are acute proof of concept studies and therefore one must be cautious about inferred long - term clinical significance , which still requires confirmation in experimental studies . adhd is a heritable and disabling disorder characterized by core cognitive and behavioural symptoms of impulsivity , hyperactivity , and inattention . it is the most prevalent neuropsychiatric childhood disorder which affects around 37% of children worldwide ( apa , 2000 ; polanczyk et al . 2007 ) . structural abnormalities in fronto - striato - circuitry ( durston et al . 2003 ) and dysfunction in catecholamine neurotransmission , specifically in na and da pathways in the prefrontal cortex ( pfc ) have been implicated in adhd ( yang et al . this leads to inefficient information processing and hypo - activation in the frontal lobes ( stahl , 2008 ) . as a result , adhd patients have significant impairments in performing working memory ( wm ) and executive function tasks ( biederman et al . 2007 ; however , if these impairments are not treated early , they can lead to significant negative life events such as drop out from education , job dismissal , criminal activities , substance abuse , and driving accidents ( barkley , 2006 ) . 2008 ) , selective na reuptake inhibitor ( snri ) atomoxetine ( strattera ) ( chamberlain et al . 2007 ; donnelly et al . 2009 ) and modafinil ( turner et al . 2003 ) , increase da and na levels in the pfc ( mehta et al . 2004 ; stahl , 2009 ; wilens , 2006 ) and alleviate cognitive impairments in adhd patients ( devito et al . 2008 ; turner et al . indeed , studies in our laboratory that use double - blind placebo - controlled designs showed that methylphenidate improves wm , cognitive flexibility , attention and response inhibition in both children and adults with adhd ( devito et al . 2008 ; ( 2008 ) used the cambridge gambling task , a test of decision making and risk taking , and showed that a single 0.5 mg / kg dose of methylphenidate reduced large bets on this task in adhd boys , who performed similarly to healthy boys without medication . in consequence , pces can effectively improve core symptoms , abnormal behaviours , self - esteem , cognition , social and family function in adhd patients ( sahakian & morein - zamir , 2007 ) . however , methylphenidate is successful in treating only about 6070% of adhd children , meaning that 30% of patients with adhd either do not respond to treatment or the drug causes adverse side - effects , such as headache , stomach pain , loss of appetite , trouble sleeping , dizziness and nausea , which precludes the use of methylphenidate . atomoxetine may be a more acceptable treatment due to its low abuse liability and minimal adverse side - effects ( heil et al . still , unlike methylphenidate which improves spatial wm ( swm ) , sustained attention , and response time in adhd patients and healthy volunteers ( elliott et al . 1997 ; turner et al . 2004b ) , atomoxetine only improves response inhibition possibly due to its selective na modulation ( chamberlain et al . 2006 , 2007 ) , and is likely to be less effective in treating the range of cognitive deficits associated with adhd . therefore , there is a need for medication with improved efficacy and reduced side - effects for adhd . furthermore , studies employing the same methodology show that modafinil also significantly improves short - term memory span , visual memory , spatial planning , and stop - signal motor inhibition in adhd adults ( turner et al . these improvements are consistent with randomized , double - blind placebo - controlled clinical trials with modafinil that demonstrate symptom reduction in adhd children and adolescents ( biederman et al . 2005 , 2006 ; greenhill et al . 2006 ; swanson et al . hence , in psychiatry ethics , developing novel pces that improve the wellbeing and quality of life for these patients meets the right to receive effective treatment that would offer them a reasonable opportunity to improve their mental condition ( bloch & green , 2008 , p. 490 ) . another important argument for developing novel pces relates to the rise of pharmacogenomics and individualized medicine that aim to combat neuropsychiatric and neurodegenerative diseases . there is growing evidence that key gene variants can change activity within specific neuronal circuits and , as a result , influence particular cognitive - affective phenomena . for example , the catecholamine - o - methyltrasferase ( comt ) gene has been shown to affect responses to comt inhibitors and to predict wm performance whereas the val polymorphism exerts a significant effect on enzyme activity and affects da - regulated pfc activity during wm tasks , and also modifies the effect of dopaminergic drugs ( e.g. the comt enzyme inhibitor tolcapone ) in the pfc ( diaz - asper et al . similarly , the therapeutic response in ad appears to be genotype - specific , with apoe-4/4 carriers being the worst responders to conventional treatments ( cacabelos , 2005 ) . however , although behavioural phenotypes and action of pces are generally complex ( diaz - asper et al . 2006 ) , both reflecting the action of multiple genes and neurotransmitters respectively , it is possible that using pharmacogenomics to develop targeted pces for particular subgroups and individual responsiveness will lead to greater efficacy and reduced side - effects . for example , turner et al . ( 2003 ) showed that a single oral dose of modafinil ( 100 mg or 200 mg ) significantly improved performance on tests of digit span , visual pattern recognition memory , spatial planning , and stop signal reaction time ( ssrt ) task , or response inhibition , in healthy volunteers . modafinil also improved the response time in tests of decision making , delayed matching to sample , and spatial planning ( mller et al . more recently , it improved accuracy in an attention - shifting task , without reaction time trade - off ( marchant et al . ( 2004 ) demonstrated that modafinil significantly reduced error rates in a long - delay visuo - spatial task and manipulation conditions , without speed - accuracy trade - off . ( 2009 ) used functional magnetic resonance imaging and examined the brain mechanism by which atomoxetine exerts its cognitive enhancing effects in healthy volunteers . they found that atomoxetine led to increased activation in the right inferior frontal gyrus ( rifg ) when participants attempted to inhibit their responses in the ssrt task . the rifg has previously been shown to be activated during inhibitory motor control ( aron et al . in contrast , methylphenidate has been shown to enhance swm performance in healthy adults ( elliott et al . ( 2000 ) showed that methylphenidate improves both performance and efficiency in the swm neural network , which includes the dorsolateral pfc and posterior parietal cortex in healthy volunteers ( owen et al . these areas have been significantly associated with wm and executive functions ( robbins et al . moreover , studies using positron emission tomography and contrasting [ c]raclopride binding , with the participants either on or off methylphenidate , have further indicated that methylphenidate influences dopaminergic function , particularly in the striatum ( wang et al . other da agonists improve wm and performance of executive tasks in healthy individuals ( mehta & riedel , 2006 ; roesch - ely et al . for example , evidence from healthy volunteers show that ampakine cx516 and cheis also lead to moderate improvements in recall and short - term memory ( wezenberg et al . in particular , pilots who took donepezil just before learning specific manoeuvres in a flight simulator outperformed a control group on tests of performance conducted 1 month later ( yesavage et al . however , the mechanism of action for improvement in attention , memory and executive function of pces still remains to be determined in many cases . for example , to exert its cognitive enhancing effects , modafinil has been shown to elevate numerous neurotransmitters including na , da and glutamate ( minzenberg & carter , 2008 ; volkow et al . evidently , as these improvements relate to neurotransmitter modulation and function ( iversen et al . 2009 ) , the effects of some pces might follow the yerkes dodson law , which explains the relationship between arousal and performance . this principle might be translated to several neurotransmitter systems where cognitive function often follows an inverted u - shaped curve , with deviations from the optimal level in either direction producing sub - optimal performance ( robbins & sahakian , 1979 ; robinson & sahakian , 2009 ) . for instance , low levels of na release engage 2-adrenergic receptors and improve executive function whereas higher levels of na release engage 1-adrenergic receptors which cause significant stress in humans and animals ( arnsten , 2000 ; finlay et al . 1995 ) and impair prefrontal functionality ( ramos & arnsten , 2007 ) . a similar u - inverted relationship is evidenced between da and wm function ( vijayraghavan et al . 2007 ) as both marked increases and decreases of da in the pfc have been associated with sub - optimal performance ( cools et al . consistent with this hypothesis , methylphenidate improves cognitive performance in individuals with greater impairment ( konrad et al . 2000 ) while guanfacine ( tenex ) , an 2-adrenergic receptor agonist , has beneficial effects on wm and attentional functions in patients with adhd , but does not improve wm or executive functions in healthy male volunteers ( mller et al . 2005 ) . thus , the effects of pharmacological substances on cognition are complex as cognition is a multifaceted construct encompassing numerous mental functions including both cold cognition ( such as attention , planning , problem solving , and response inhibition ) and hot cognition ( such as risky decision making ; roiser et al . a recent study by farah et al . ( 2009 ) showed that the mixed amphetamine salts , adderall , licensed for the treatment of adhd , enhanced performance on convergent tasks of creativity for lower - performing individuals and either impaired or did not change it for higher - performing individuals . these results on improvement and impairment on higher cognitive function with pces raises the issue of what we mean by a general term enhancement. as healthy adults fall into a wide spectrum of normality , some individuals may be improved by a pce drug while others remain unchanged or are even impaired ( randall et al . furthermore , there is as yet no robust empirical research to demonstrate that pces have effects on divergent thinking in healthy people . the above results demonstrate the potential of pces to enhance certain cognitive domains in healthy adults . therefore , attitudes towards their use by the general population need to be considered . in the next section , we focus our discussion on current and future trends of the use of pces by healthy people . in the past few years there has been an unprecedented rise in the use of pces among healthy individuals for cognitive enhancement . cognitive enhancement can be defined as the amplification or extension of core capacities of the mind through improvement or augmentation of internal and external information processing systems ( bostrom & roache , unpublished data ) . healthy university students ( desantis & hane , 2010 ) and academics ( sahakian & morein - zamir , 2007 ) have been using pces to improve their cognitive function . more specifically , students are taking pces to improve academic performance ( rabiner et al . 2009 ) and are framing their actions as both physically harmless and morally acceptable ( desantis & hane , 2010 ) . for example , in the usa , 16% of college students ( babcock & byrne , 2000 ) and 8% of undergraduates reported having illicitly obtained and used prescription stimulants ( hall et al . furthermore , a 2005 survey by the us national institute on drug abuse ( nida , 2005 ) found that 2.5% of 13- to 14-yr - olds , 3.4% of 15- to 16-yr - olds and 5.1% of 17- to 18-yr - olds abused methylphenidate . in 2009 , the figures for these groups were 1.8% , 3.6% and 2.1% , respectively ( nida , 2009 ) . presumably , these young people are obtaining stimulant drugs from others who have prescriptions or purchasing them via the internet or street dealers . currently , the global market share of modafinil is more than us$700 million per year ( norman & berger , 2008 ) . consistent with greely 's ( 2006 ) claim , that healthy physicians on call , students and academics are increasingly using pces to enhance cognitive abilities , it is estimated that around 90% of modafinil is predominantly used off - label by healthy , non - sleep - deprived individuals ( baranski et al . in contrast , beta - blockers that are prescribed to reduce anxiety in clinical patients have been used by musicians to dampen physiological tremors in order to improve their performances on stage ( tindal , 2004 ) . in the uk , a newspaper survey of 1000 students showed that 1 in 10 were taking prescription drugs for cognitive enhancement ( lennard , 2009 ) . in england , prescription rates of stimulants have been rising steadily from 220000 in 1998 to 418300 in 2004 ( niyadurupola , 2008 ) . in 2008 , the journal nature conducted a poll about the use of pces by healthy academics , in which 1400 scientists from 60 different countries responded ( maher , 2008 ) . one in five respondents used drugs for cognitive enhancement , with 52% of them obtaining the drug by prescription , while 34% obtained the drug via the internet and 14% through their pharmacy . the most popular drug was methylphenidate , with 62% of users ; 44% reported taking modafinil mainly to improve concentration , and 15% reported taking beta - blockers for anxiety . of all respondents , 96% thought that people with neuropsychiatric disorders should be given cognitive enhancing drugs . in contrast , 86% of respondents thought that healthy children under the age of 16 yr should be restricted from taking pce drugs . although some of these data were not rigorously collected , they nonetheless suggest the increased use of pces among healthy individuals . their widespread use is not surprising given that small percentage increments in performance can lead to significant improvements in functional outcome . indeed , a 10% improvement in memory score could lead to an improvement in an a - level grade or degree class ( academy of medical sciences , 2008 , p. 150 ) . nevertheless , the increase in lifestyle use of pces by healthy people raises numerous ethical issues that inform the growing field of neuroethics . neuroethics is the study of the ethical , legal and social questions that arise when scientific findings about the brain are carried into medical practice , legal interpretations and health and social policy ( marcus , 2002 ) . as such , modifying our inherent self , character and individuality through pce drugs has important implications for society . for these reasons , their lifestyle use has prompted a significant interest both in the media and the public ( coveney et al . there is a concern that pces will threaten our notion of personhood and will dampen essential characteristics of what it means to be human ( farah et al . 2004 ; president 's council on bioethics , 2003 ) . as a consequence , enhancing the brain and higher cognitive processes demands strong ethical considerations and a practical policy framework . to address this , we have argued elsewhere that before pce drugs are prescribed to healthy people , their long - term safety , side - effects and their effectiveness must be tested to provide important facts necessary for further decision making about their regulation ( sahakian & morein - zamir , 2010 ) . moreover , we have engaged with the media about the need to establish regulations for the use of pce drugs by healthy people ( mohamed & sahakian , 2010 ) . in nature , we emphasized the need to ensure their safe use by healthy people ( greely et al . 2008 ) while in science we advocated that ethical considerations in regard to societal issues associated with the use of pces by healthy people should be part of neuroethical training within university neuroscience programmes ( sahakian & morein - zamir , 2009 ) . since pces improve those with low cognitive performance ( robbins & sahakian , 1979 ) , it might be possible to mitigate the adverse environmental effects , such as poverty , on the brain and cognition through their use . it may also be that some healthy people actually have undiagnosed attentional or other problems and are actually self - medicating with drugs such as ritalin . furthermore , even healthy adults , who normally function well , are not always performing optimally due to sleep deprivation , jet lag or other stressors , and some might need to perform at their best possible level on every occasion ( e.g. surgeons , air traffic controllers ) . in addition , pces might enable us to perform better in other competitive or life threatening situations . for instance , psychostimulants have been employed to boost cognition in soldiers in combat ( caldwell et al . pces have also been demonstrated to improve performance in shift workers ( ballon & feifel , 2006 ) , pilots ( caldwell , 2001 ) and school pupils with adhd ( trout et al . recently , the us defence advanced research project agency ( darpa , 2007 ) introduced the augmented cognition programme to enhance soldiers memory and cognition through technology when under conditions of interrupted sleep and stress . however , if proven to be safe , pces may be preferred for cost - effectiveness when compared to other methods of enhancement such as expensive technology . in contrast , the disadvantages of using pces include the potential harms and long - term side - effects that they might have in healthy people , particularly in adolescents where the brain is still in development . there are strong safety concerns , especially in the absence of informative data , for healthy individuals as the risk of adverse side - effects might outweigh the beneficial effects of pce drugs . the abuse liability of some of the pces such as methylphenidate is also a concern . a recent study showed that modafinil blocked da transporters and increased da in the caudate , putamen and nucleus accumbens in healthy human brain ( volkow et al . 2009 ) , which are areas in a network known to be involved in drug - seeking behaviour and addiction ( volkow & li , 2004 ) . this indicates the need for awareness about the risks involved in pce use among healthy people and shows that a full ethical consideration of their use is required . to date , there have been no randomized psychopharmacological trials investigating the long - term effects of pce drugs on healthy people . there is still a further safety concern about the risks of purchasing substances advertised as pces over the internet ( forman et al . as these drugs are not prescribed by a qualified doctor , they might not be suitable for some people . for instance , contra - indications of atomoxetine and modafinil include heart problems and hepatic impairments ( british national formulary , 2010 ) drug interactions which could be dangerous in some cases . with regard to personal autonomy , there are ethical concerns about healthy people being coerced or even forced into using a pce . society might force people to take psychoactive agents in order to perform better or to be in a particular mental state . for example , authorities in the usa ordered a mentally ill inmate in criminal proceedings to take psychotropic medication to improve his competence to stand trial and be executed ( boire & ruiz - sierra , 2003 ; randall , 2004 ) . there is also a considerable potential for indirect coercion resulting from a highly demanding 24/7 society where people feel compelled to take pces in order to meet social or workplace demands . healthy people may resort to self - medication for inadequate sleep or over - exertion at work . for example , 33% of respondents in maher 's ( 2008 ) poll indicated that they would feel pressure to give pce drugs to their children if other children at school were taking them . however , the use of pces to enhance cognition is one solution to improving the individual and society . indeed , we have argued elsewhere that there are other methods of boosting cognition , including education and exercise ( sahakian & morein - zamir , 2010 ) . for instance , physical exercise can improve learning and memory ( creer et al . we might be able to effectively and safely enhance cognition and well - being in society . another argument against their use is that they might further exacerbate the ever - growing disparity and inequality in society , especially if only the wealthy can access them . equally , is it morally justified to use pces during exams , and does it give the user unfair advantage over those who are equally capable but not cognitively enhanced with drugs ? many universities as yet have no formal policy about the use of pces during exams . if pces become easily accessible in the future , will society consider their use as cheating or will they equate them to having a caffeine boost from coffee ? is it possible that once pce drugs are widely available we might run the risk of becoming a homogeneous society ? could our perception of ourselves change from being human to being mechanistic beings with a modicum of emotion ? are we going to be over - enhanced only to be plagued by unwanted memories ? will using pces outdate important human virtues such as hard work and reflection and make us unable to take credit for our minds achievements ? the advantages and disadvantages of using pces have to be evaluated carefully . with regards to fairness , enhancing cognition might lead to dramatic social benefits by reducing natural inequality and promoting social justice ( savulescu , 2006 ) . this is because increasing cognitive ability on an individual level could have dramatic and positive effects on society and the economy as a whole ( bostrom , 2008 ) . for instance , a 3% population - wide increase in iq would reduce poverty rates by 25% ( weiss , 1998 ) , and would lead to an annual economic gain of us$165195 billion and up to 1.5% gdp growth ( salkever , 2005 ; schwartz , 1994 ) . however , if healthy people take pces to gain a competitive edge but fail to see any difference in the long - term or notice possible impairments observed in high - functioning adults ( mattay et al . hence , determining who can use pces and under what circumstances involves complex decision making and ethical judgements . thus , how neuroscientific discoveries impact on society has given rise to an enormous interest in the field of neuroethics , including the foundation of the neuroethics society ( http://www.neuroethicssociety.org ) which advocates further research on ethical questions that are yet to be answered . for instance , what are the possible long - term harms of using pce drugs in healthy people , particularly in the developing brain ? what are the implications of developments in pharmacogenomics ? without formal regulation of their use , healthy people can purchase pces via the internet , with all the inherent dangers in doing so . how would such easy access affect widespread use of these drugs by healthy young and elderly people and also impact on society ? how would we , as neuropsychopharmacologists , react if we discover that our colleagues or our children 's friends are taking pces ? how should governments react ? they also clearly indicate the need to engage in discussion with the public about the social and ethical implications of the use of pces by healthy individuals ( morein - zamir & sahakian , 2009 ; ringach , 2009 ) . for this to happen , neuropsychopharmacologists need to integrate experimental results within a neuroethical framework . in order to do this , they need to innovatively work together with social scientists , philosophers , and ethicists ( morein - zamir & sahakian , 2009 ; sahakian & morein - zamir , 2009 , 2010 ) . 2004 ) and puts them in a leading position to engage policy makers and a broad group of stakeholders , including the general public . this will ensure that technological advances in neuroscience are put to maximal benefit and minimal harm . pces have the potential to ameliorate cognitive dysfunction and to provide important clinical benefits for patients . further development of more effective pces with fewer side - effects , in addition to neuroprotective agents for patients with neurodegenerative diseases such as ad , is clearly worthy of pursuit . however , their long - term cognitive enhancing potential as well as their side - effects in healthy people needs to be rigorously determined . currently , the unprecedented rise of pce use among the healthy raises numerous ethical issues . scientists need to work together with social scientists , philosophers , ethicists , policy makers , and teachers to actively discuss the ethical consequences of pce usage . finally , the use of pces to enhance cognition is one solution to improving the individual and society . however , this does not preclude other means of enhancing cognition such as education and exercise ( beddington et al .

pharmacological cognitive enhancers ( pces ) are used to improve cognitive functions , such as attention , learning , memory and planning in patients with impairments in cognition resulting from traumatic brain injury ( tbi ) or from neuropsychiatric disorders such as alzheimer 's disease ( ad ) , mild cognitive impairment , schizophrenia , and attention deficit hyperactivity disorder ( adhd ) . moreover , pces have been shown to improve cognition in healthy volunteers with no psychiatric disorders . this article describes the rationale behind the need for their use in neuropsychiatric patients and illustrates how pces can ameliorate cognitive impairments , improve quality of life and wellbeing , and therefore reduce the economic burden associated with these disorders . we also describe evidence that pces are being used as cognitive enhancers by healthy people . crucially , as the lifestyle use of these drugs becomes very popular in the healthy population , a final aim is to present an overview of the current and future neuroethical considerations of enhancing the healthy brain . as information regarding their actual use , benefits and harms in various healthy populations is currently lacking , we propose research that aims to obtain relevant empirical data , monitor the short- and long - term effectiveness and side - effects , and initiate accurate surveys to determine current patterns and quantity of usage of pce drugs by healthy people . furthermore , in order to instigate a dialogue between neuroethics and neuropsychopharmacology , we urge scientists to explore and communicate the social and ethical implications of their research to the public . finally , we discuss and highlight other means of enhancing cognition in both patients and healthy adults , including education and physical exercise .

Pharmacological cognitive enhancers (PCEs) Pharmacogenomics PCEs improve cognition in healthy individuals Neurotransmitter modulation of cognition Lifestyle use of PCEs by healthy individuals Neuroethical issues in cognitive enhancement Public engagement in neuroscience Conclusions

PMC2762119

to t cells , they lack receptor diversity yet demonstrate remarkable plasticity in response to both infectious and noninfectious stimuli . as an extension of their dichotomous nature , t cells have been held liable for both instigating over - zealous immune responses [ 13 ] as well as reigning in inflammation by controlling activated macrophages and promoting wound healing [ 46 ] . in humans , the main subset of peripheral blood t cells usually comprises between 1 and 5% of circulating t cells and bears the canonical v9v2 t cell receptor . these v9v2 t cells are unique to primates , and they respond to endogenous and exogenous stress - induced molecules that are nonpeptidic in nature and preclude the requirement for processing or presentation by classical mhc molecules [ 79 ] . isopentylpyrophosphate ( ipp ) is the prototypical phosphoantigen recognized by v9v2 t cells that was first isolated from lysates of mycobacteria . it was later determined to be a metabolite in the mevalonate pathway in all eukaryotic and some prokaryotic cells , including staphylococcus aureus [ 10 , 11 ] . during the initial phase of infection with low bacterial inocula of s. aureus , v9v2 t cells are activated by endogenous mevalonate metabolites through the accumulation and dephosphorylation of the hydroxymethylglutaryl - coenzyme a reductase , the rate - limiting enzyme of the mevalonate pathway . therefore , primates have evolved the ability to readily respond to bacterial infection as well as endogenous damage by sensing the dysregulation of the mevalonate pathway , which may be considered a stress - associated alarmin for these primordial lymphocytes . we have shown that stimulating the small pool of human t cells present in pbmc with ipp markedly augmented the early proinflammatory response to toxic shock syndrome toxin-1 ( tsst-1 ) . tsst-1 is a staphylococcal superantigen that exerts a potent inflammatory response by bridging v2 specific t cell receptors with conserved regions of the mhc class ii molecules of antigen presenting cells ( apcs ) outside the peptide - binding groove . demonstrated the presence of specific v t cell signatures of various superantigenic toxins , including tsst-1 , in the blood of patients with staphylococcal tss . we previously demonstrated that ipp - activated t cells augment the levels of proinflammatory cytokines within the first 6 hours post - tsst-1 treatment , thereby changing the kinetics and magnitude of the immune response to the superantigen . we have also shown that high mobility group box protein-1 ( hmgb-1 ) is released following tsst-1 stimulation . hmgb-1 is a highly conserved nonhistone dna - binding protein found in virtually all nucleated cells and is regarded as being the late mediator of sepsis . activated monocytes secrete hmgb-1 after exposure to endotoxin , and it is detectable in the serum of septic patients within 24 hours . importantly , neutralizing the effects of hmgb-1 at this late time point , either by anti - hmgb-1 antibodies or antagonists , rescued mice from sepsis - induced mortality . in contrast to endotoxin , which activates only macrophages to secrete hmgb-1 , both t cells and macrophages participate in the nuclear translocation and subsequent release of hmgb-1 into the extracellular environment following tsst-1 stimulation . the first aim of the present study was to evaluate the influence exerted by ipp - primed v9v2 t cells on the change in costimulatory molecule expression that could explain their ability to enhance the immune response to tsst-1 . the second objective was to determine the consequence of t cell mediated immune modulation on the expression and secretion of hmgb-1 . recombinant tsst-1 was purified from culture supernatants of s. aureus strain rn4220 previously transformed to carry the tst gene , using both preparative isoelectric focusing and chromatofocusing . toxin purity was assessed by silver staining after sodium dodecyl sulfate - polyacrylamide gel electrophoresis on 14% acrylamide gels , and lps activity was undetectable by the limulus amoebocyte lysate gelation ( sensitivity limit , 10 pg / ml ) . fresh human peripheral blood mononuclear cells ( pbmc ) from healthy donors were obtained by ficoll - paque plus ( amersham biosciences corp . , piscataway , nj ) density centrifugation , and cultured in 96-well u - bottom plates at 1.5 10 cells / ml in complete culture medium consisting of rpmi 1640 ( stemcell technologies inc . , vancouver , bc , canada ) , 10% heat - inactivated fetal bovine serum ( hyclone laboratories inc . , logan , ut ) , 2 mm l - glutamine ( stemcell ) , 25 mm hepes buffer ( stemcell ) , and 2 ug / ml of polymyxin b sulphate ( sigma - aldrich corp . , st . depletion of t cells was accomplished by immunomagnetic positive cell selection with an anti- tcr mab ( clone immu510 , bd biosciences pharmingen inc . , san diego , ca ) conjugated to an antidextran mab tetramer ( stemcell ) , which left the rest of the pbmc untouched , using the stemsep protocol ( stemcell ) . purity of the t cell - depleted pbmc was analyzed by flow cytometry using phycoerythrin-(pe- ) conjugated anti - v2 antibody ( bd biosciences pharmingen ) . figure 1 shows the population of v9v2 t cells in pbmcs before ( which made up 1.5% of live cells ) and after depletion ( 0.1% ) . pbmcs with or without t cell depletion were treated with either 45 m ipp ( sigma - aldrich ) or left untreated in complete growth medium for 1618 hours ( overnight ) at 37c , 5% co2 prior to treatment with 1 nm tsst-1 . for secreted hmgb-1 detection , culture supernatants were microcentrifuged at 800 g for 5 minutes and frozen at 70c until analysis . surface expressed hmgb-1 was analyzed by flow cytometry ( facscalibur flow cytometry system , bd biosciences pharmingen ) using the anti - hmgb-1 antibody and secondary alexa488-conjugated antibody ( as described below ) . costimulatory molecule cell surface expression was performed by incubating cells at 4c ( protected from light ) for 30 minutes with the following fluorescently - conjugated antibodies : apc antihuman hla - dr ( ln3 ) , pe - cy5 anti - human cd86 ( b7 - 2 ) ( ebioscience , inc ) , fitc anti - cd40 ( 5c3 ) , pe - cy5 anti - cd80 ( b7 - 1 ) , pe - cy5 anti - cd28 ( cd28.2 ) , and apc anti - cd25 ( m - a251 ) ( bd biosciences , pharmingen ) . cells were subsequently washed , resuspended in buffer containing 2 mm edta ( to facilitate detachment of adherent cells ) , and transferred to facs to analyze by flow cytometry ( a minimum of 10 000 events were collected ) . surface expression of hmgb-1 on pbmc by fluorescent microscopy has been previously described in detail . in brief , pbmcs were cultured ( 37c , 5% co2 ) directly on sterile cover slips to allow attachment of adherent cells overnight before stimulation with 1 nm of tsst-1 . for hmgb-1 expression , cells were incubated with rabbit anti - human hmgb-1 igg as previously described , diluted in blocking buffer ( pbs , 3% fbs ) , followed by a secondary alexa488-conjugated goat antirabbit igg antibody . fluorescently labelled pbmcs were then stained with hoechst 3342 nuclear dye ( molecular probes ) according to manufacturer 's directions , and mounted on slides using prolong anti - fade reagent ( molecular probes ) . cells were visualized with an axioplan ii fluorescence microscope equipped with a ccd camera using northern eclipse software ( epix ) for acquisition of images . images were taken with the 63 x oil immersion objective lens , and adobe photoshop 6.0 software was used for image layout . pbmc culture supernatants were concentrated ( ~10-fold ) from the original volume of 1.5 ml using amicon ultra centrifugal filters with a molecular weight cutoff of 10 kda ( millipore ) . the supernatants then had been further prepared using the sds - page clean - up kit ( amersham ) according to the manufacturer 's directions prior to running on a 12% polyacrylamide gel . western blotting was performed by semidry transfer of proteins ( trans - blot sd semi - dry electrophoretic transfer cell , biorad ) onto an immobilon - p pvdf membrane ( millipore ) which was blocked for 1 hour at room temperature with 1% bsa , 0.5% tween in tbs prior to overnight incubation ( at 4c ) with rabbit polyclonal anti - hmgb-1 antibody . the membrane was subsequently incubated with anti - rabbit igg horse - radish - peroxidase-(hrp- ) conjugated secondary antibody for 1 hour at room temperature on a shaker . hmgb-1 detection was performed using super signal substrate ( pierce ) and developed as well as analyzed using the alpha innotech 3400 gel documentation system ( alpha innotech ) . student t - tests ( one - tail ) were used to assess the significance of differences in hmgb-1 and co - stimulatory molecule expression between ipp treated and untreated pbmc at different times post - tsst-1 stimulation . the effects of ipp over time on co - stimulatory molecule expression by apc ( cd40 , cd80 , cd86 , hla - dr ) and t cells ( cd25 , cd28 ) were further examined by 2-way anova . differences were considered significant if the probability of the null hypothesis was less than five percent ( p < .05 ) . following overnight treatment with ipp , cd40 expression was upregulated in comparison to those that were untreated ( ipp + tsst-1 versus tsst-1 alone ; p < .05 , student 's t - test ) ( figure 2(a ) ) . the elevated cd40 expression was observed up to 6 hours postsuperantigen stimulation , after which time cd40 was downregulated or internalized . we previously demonstrated that cd40l ( cd154 ) is expressed on t cells within 3 hours following exposure to tsst-1 , therefore , the down - regulation of cd40 beyond 6 hours is likely a consequence of cd40 binding to cd40l on tsst-1-activated t cells . we verified that up - regulation of cd40 expression in populations exposed to ipp was solely attributed to the presence of activated t cells , since removal of t cells completely abrogated the effect of ipp ( figure 2(b ) ) . the effect of ipp - activated t cells over time on cd40 expression following tsst-1 stimulation was further verified using 2-way anova ( ipp x time interaction , p < .05 ) . in contrast to cd40 , the expression of cd86 was down - regulated early in ipp treated pbmc at 3 hours following tsst-1 stimulation ( ipp + tsst-1 versus tsst-1 alone ; p < 0.05 , student 's t - test ) ( figure 2(a ) ) . again , this effect was abrogated following depletion of t cells ( figure 2(b ) ) . two - way anova confirmed an interaction effect ( ipp x time interaction , p < .05 ) . there was a similar trend for decreased expression of hla - dr ( the mhc class ii molecule to which tsst-1 binds on apc ) and cd80 by ipp treatment , but the differences were not statistically significant . in contrast to the direct affect of ipp on monocyte function , there was little notable direct influence on the activation state of t cells as evaluated by the expression of cd25 or cd28 , which remained largely unchanged in response to ipp pretreatment ( figure 3 ) . the upregulation of cd25 on t cells at 24 hours was solely dependent on tsst-1 stimulation regardless of ipp , as shown by the comparison of ipp + tsst-1 versus ipp alone ( p = .001 , student 's t - test , figure 3 ) . two - way anova confirmed this effect ( tsst-1 effect , p < .01 ; tsst-1 x time interaction , p < .005 ) . established that monocytes that had become adherent export hmgb-1 to the cell surface where it serves a function for process extension , migration , and cell - cell interaction . extracellular cell - surface expressed hmgb-1 has been called amphoterin to differentiate it from intracellular hmgb-1 found in the nucleus . given the direct influence of t cells on the activation , maturation , and regulation of cells of monocytic lineage [ 20 , 21 ] , we wanted to determine how ipp stimulation would influence the surface manifestation of hmgb-1 on cultured monocytes present in pbmc . figure 4(a ) shows that resting adherent cells have hmgb-1 ( stained in green ) localized at focal points around the perimeter in vesicle - like pockets ( top left corner ; rpmi ) . t cells , which are nonadherent cells in suspension ( stained in red ) , do not express hmgb-1 on the surface in this manner . adherent monocytes bearing surface expressed hmgb-1 subsequently released it following tsst-1 stimulation ( figure 4(a ) ; top right panel ) . overnight treatment with ipp alone also led to a change in extracellular hmgb-1 expression in pbmc cultures ( figure 4(a ) ; bottom left panel ) . there was an observable modification on both the morphological appearance of macrophages as well as the distribution of hmgb-1 around the perimeter , which became less concentrated in vesicle - like foci and more dispersed . overnight incubation of pbmcs with ipp followed by tsst-1 stimulation resulted in significantly more hmgb-1 being released into the extracellular environment compared to tsst-1 alone ( figure 4(a ) , upper and lower right panel ) . this change in surface expressed hmgb-1 was assessed by flow cytometry , which enabled us to both confirm and quantify the release of hmgb-1 from differentiated monocytes following tsst-1 stimulation . figure 4(b ) displays the difference between the various treatment conditions on the loss of cell - surface expressed hmgb-1 assessed by flow cytometry . ipp - pretreated pbmcs that were subsequently stimulated with tsst-1 released more hmgb-1 from the cell surface ( i.e. , less hmgb-1 expression on the cell surface ) than those cells that were left untreated with ipp prior to superantigen stimulation ( ipp + tsst-1 versus tsst-1 alone , p < .05 , paired student 's t - test ) . the actual secretion of hmgb-1 into the extracellular milieu from tsst-1 stimulated pbmc was further verified by western blot of cell culture supernatants ( figure 4(c ) ) , which again substantiated that ipp pretreatment followed by tsst-1 stimulation led to its greater release than pbmc stimulated with tsst-1 without ipp - activation of t cells ( ipp + tsst-1 versus tsst-1 alone , p < .05 , paired student 's t - test ) . overnight treatment with ipp resulted in a notable up - regulation of cd40 on monocytes in the presence of t cells . we previously demonstrated that impeding the cd40-cd40l interaction with cd40 monoclonal antibodies ( but not the cd86 or cd80 interaction ) completely blocks tsst-1-induced inflammation . the early up - regulation of cd40 on monocytes by ipp - primed t cells in the current study provides a basis for the prior observation that tnf and ifn levels increased 3-to 4-fold during the early phase of the immune response to tsst-1 in pbmc containing activated t cells . this study also demonstrates that the exaggerated response to tsst-1 by activated t cells is through the direct modulation of the responsiveness of monocytes and not t cells since we found that the upregulation of cd25 on t cells was affected only by stimulation with tsst-1 and not ipp . the regulation of cd40 expression by activated t cells appears to parallel the mechanism of linking innate to adaptive immunity by glycolipid responsive v14 + nk t cells stimulated with -galactosylceramide . it was found that the rapid release of tnf and ifn to ovalalbumin , acquisition of cd4 + and cd8 + t cell immunity , and maturation of dendritic cells were all dependent on the cd40-cd40l interaction induced by the priming of v14 + nk t cells by -galactosylceramide . in contrast to cd40 , cd86 expression was found to be reduced in pbmc treated with ipp overnight . this monocytic phenotype may be reflective of a state described as immature dendritic cells in which cycling of the hla - dr molecule is rapid and levels of cd86 are relatively low . alarmins influence the ability of apc to effectively stimulate cells of adaptive immunity , and the cd40-cd40l interaction likely plays a pivotal role in this process . this scenario is in agreement with recent demonstrations that t cells induce early dendritic cell maturation and share an intimate relationship with apc [ 21 , 24 , 25 ] . further provided evidence that human t cells are activated by microbial mevalonate metabolites and interact with monocytes and local macrophages to drive the acute inflammation in bacterial infections . . it should be noted , however , that these studies primarily used either expanded t cell lines or a large number of purified t cells in coculture conditions . the conditions used in this study may be more reflective of the physiological state in which a small number of t cells present in peripheral blood would be able to immediately respond to their stress - associated antigens , such as ipp . this early event would preclude the possibility of any expansion of t cells , but it would lead to the efficient priming of apc . this study also provides the first demonstration that ipp - primed human peripheral blood t cells influence the secretion of hmgb-1 by macrophages . this finding is similar to the work by semino et al . who demonstrated that nk cells reciprocally activate autologous dendritic cells , and this interaction was also mediated through the secretion of hmgb-1 . it is feasible that the enhanced hmgb-1 secretion observed in cells containing activated t cells could be in response to an increase in apoptosis due to the fact that ipp - stimulated t cells are particularly susceptible to activation - induced cell death [ 29 , 30 ] as a self - limiting precaution . during apoptosis , hmgb-1 is normally tightly bound to nucleosomes as opposed to necrosis where hmgb-1 is released from cells one of the main ligands for extracellular hmgb-1 is the receptor for advanced glycation end ( rage ) products , a member of the ig superfamily . the expression of rage increases following activation on both dendritic cells and antigen stimulated t cells . the release of hmgb-1 and its subsequent binding to rage is required for the clonal expansion , survival , and functional polarization of naive t cells . both dendritic cell maturation and th1 polarization are events that have been attributed to the activity of t cells and hmgb-1 , respectively [ 21 , 3436 ] . our findings connect these two previously unrelated concepts and brings forth the suggestion that t cells mediate this effect and link innate and adaptive immunity by influencing the early upregulation of cd40 expression and release of hmgb-1 from apc , thereby priming the immune response to subsequent stimuli .

t cells play an important role in regulating the immune response to stress stimuli ; however , the mean by which these innate lymphocytes fulfill this function remains poorly defined . the main subset of human peripheral blood t cells responds to nonpeptidic antigens , such as isopentylpyrophosphate ( ipp ) , a metabolite in the mevalonate pathway for both eukaryote and prokaryote cells . ipp - primed t cells significantly augment the inflammatory response mediated by monocytes and t cells to tsst-1 , the staphylococcal superantigen that is the major causative agent of toxic shock syndrome . here we show that the small pool of activated peripheral t cells induces an early upregulation of cd40 on monocytes and the local release of high mobility group box-1 ( hmgb-1 ) , the molecule designated as the late mediator of systemic inflammation . this finding provides a new basis for how t cells may serve as influential modulators of both endogenous and exogenous stress stimuli .

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

PMC3531208

transfer rnas ( trnas ) play essential roles in cell viability . beyond their major function in translating the genetic code , trnas are implicated in many other processes such as viral replication , amino acid biosynthesis or cell wall remodeling ( 13 ) . eukaryotic genomes encode a complex population of trna genes and the expression of trna species is subject to a tight regulation in particular at the transcriptional level . the development and cell differentiation of tissues may be affected by the steady - state levels of certain trnas [ e.g. ( 4,5 ) ] . the existence of trna isodecoders , i.e. trnas sharing the same anticodon but having distinct body sequences , has also the potential to play important regulatory roles ( 6,7 ) . another major observation is that subcellular trna trafficking pathways within the cell , for instance between the cytosol and the nucleus or the cytosol and mitochondria , play a role in rna quality control , stress response or organelle biogenesis . finally , over the past 5 years , deep sequencing approaches have revealed the existence of new small non - coding rnas originating from trnas and are thus called trfs ( for trna - derived rna fragments ) . some of these trfs were shown to be induced in response to stress , during aging or to be involved in translation inhibition ( 8) . the number of novel functions played by trnas or trna - derived fragments is increasing rapidly and the above list is not exhaustive . this is likely the visible part of the iceberg and further studies will necessitate easy access to the most accurate and complete set of data concerning trna gene populations of given eukaryotic organisms , as well as to relevant biological information such as upstream promoter regions , intron sequences , mitochondrial import or trna - related enzymes . as photosynthetic organisms possess three compartments encoding genetic information ( i.e. the nucleus , the chloroplast and the mitochondrion ) , they represent the most intricate and thus interesting models to obtain an integrative view of the trna gene population present within a eukaryotic cell in terms of gene content , organization , expression and function . in addition , the primary endosymbiosis of a cyanobacteria in a heterotrophic protist leading to the present - day chloroplast and the existence of secondary and tertiary endosymbiosis events leading to the great diversity of photosynthetic eukaryotes from algae to land plants represent landmark evolutionary events ( 9,10 ) that are worth to be analyzed at the trna gene level . we thus decided to focus on trna genes from photosynthetic organisms . in 2000 , the first complete sequence of a plant genome , that of arabidopsis thaliana , was published ( 11 ) . since then , with the completion of several plant and algal genomes , we now have the opportunity to study their complete trna gene sets . indeed , such an analysis was recently achieved for five flowering plants ( 12 ) , but the data were not accessible online . so far , three major trna databases are available : ( i ) the transfer rna database ( trnadb ; http://trnadb.bioinf.uni-leipzig ) ( 13 ) , ( ii ) the trna gene database curated by experts ( trnadb - ce ; http://trna.nagahama-i-bio.ac.jp ) ( 14 ) and ( iii ) the genomic trna database ( gtrnadb ; http://gtrnadb.ucsc.edu/ ) ( 15 ) . the first database , trnadb , is a restructured version of the first compilation of trna and trna gene sequences ( 16 ) and contains > 12 000 trna gene sequences from 577 organisms . this database is the only one that also provides 623 trna sequences from 104 organisms and thus offers interesting information on nucleotide modifications . however , concerning higher plants , only few sequences are available . for example , only 247 nuclear trna gene sequences mostly from a. thaliana are annotated and no full nuclear genome has been analyzed . in the second database , trnadb - ce , in particular , more than half of the sequences come from metagenome analyses of microorganisms from different environmental samples . this reliable database represents a nice tool to use trna gene sequences as genus - specific markers and study microbial population . trnadb - ce also includes trna genes retrieved from 121 complete plastidial genomes , but nuclear trna genes from only two higher plant species ( a. thaliana and oryza sativa ) are given . neither mitochondrial trna genes nor nuclear trna genes from other photosynthetic eukaryotes can be retrieved . the third database , gtrnadb , compiles trna gene sequences from various complete genomes thanks to the powerful trnascan - se program ( 17 ) and 11 land plant genomes were analyzed . as mentioned by the authors , the database is not curated which results in the occurrence of high numbers of errors ( e.g. it provides 639 trna gene for the brachypodium distachyon or 738 for o. sativa while the accurate numbers are 479 and 516 , respectively ) and no trna genes from other types of photosynthetic eukaryotes are available . however , none of them is dedicated to photosynthetic organisms and only very partial sets of data on plant or algal trna genes are available through the three web interfaces . here , the plantrna database brings together the information from 11 eukaryotes representative of evolutionary distinct branches of the photosynthetic lineage including brown and green algae , glaucophytes , bryophytes , flowering plants and diatoms . more than 4350 manually curated sequences of trna genes encoded by the nuclear , plastidial or mitochondrial genomes of these 11 species are accessible through the website . biological information relevant to trna biology ( e.g. intron sequences , flanking sequences controlling trna gene expression , mitochondrial trna import or trna - related enzyme genes ) is also provided and will be presented below . the organisms were selected based on two criteria : ( i ) the quality of their genomes annotation and ( ii ) their representativeness of evolutionary divergent branches of the photosynthetic lineage ( figure 1 ) . from the manually curated lists of trna genes that we recently extracted from the genomes of five angiosperms ( a. thaliana , o. sativa , populus trichocarpa , medicago truncatula and b. distachyon ) and one green alga ( chlamydomonas reinhardtii ) ( 12 ) , we retrieved trna genes from the genomes of six other photosynthetic organisms , namely , another green alga ( ostreococcus tauri ) , a glaucophyte ( cyanophora paradoxa ) , a brown alga ( ectocarpus siliculosus ) and a pennate diatom ( phaeodactylum tricornutum ) . recently , the release of assembly v5 of the c. reinhardtii nuclear genome was available and the trna gene data were updated . most sources of genome sequences are cited in ( 12 ) or available at http://bioinformatics.psb.ugent.be/webtools/bogas/ or http://www.phytozome.net/ ( 18 ) . whole nuclear , plastidial and mitochondrial genomes were scanned by trnascan - se ( 17 ) and then manually annotated as described in ( 12 ) . for each trna gene , the linear secondary structure as well as biological information are given . this includes ( i ) 5- and 3-flanking sequences that are involved in the control of gene expression or on polyt termination sequences , ( ii ) a and b boxes involved in tfiiic transcription factor binding and ( iii ) intron sequences . mitochondrial trna import is a widespread process , in particular in the plant kingdom ( 19,20 ) . it is thus relevant , either based on experimental data or on prediction to provide the scientific community with the mitochondrial import status of nuclear - encoded trna species . second , information on the population of enzymes related to trna biogenesis and function is given . this is particularly true for a. thaliana where > 150 enzymes were identified [ e.g. ( 2123 ) ] . in addition , even though the annotation of whole genomes is still largely incomplete , we also provide information on trna - related enzyme genes for six of the other photosynthetic species ( including bryophyte , green and brown algae and diatom ) present in the plantrna database . this information includes accession numbers corresponding to genes encoding enzymes involved in maturation steps such as 5 and 3 processing , cca addition and genes coding for aminoacyl - trna synthetases . due to the importance of the dual - targeting phenomenon for proteins involved in translation in plants , we also put strong effort in providing the subcellular localization of these enzymes . the phylogenetic tree was constructed with the full - length 18s rrna gene sequences using the neighbor - joining method . the presence of intron - containing trna genes , of genomes encoding a trna and the occurrence of trna mitochondrial import are reported . minus 5 = a , e , mi , v , w ; minus 4 = q , h , mi , f ; 8 = r , e , q , me , p , s , t , w , y. the one - letter code for amino acids is used . the phylogenetic tree was constructed with the full - length 18s rrna gene sequences using the neighbor - joining method . the presence of intron - containing trna genes , of genomes encoding a trna and the occurrence of trna mitochondrial import are reported . minus 5 = a , e , mi , v , w ; minus 4 = q , h , mi , f ; 8 = r , e , q , me , p , s , t , w , y. the one - letter code for amino acids is used . all sequences and biological datasets are stored in a database implemented in mysql version 5 ( http://dev.mysql.com ) . the querying of underlying sql database is implemented using java servlets running on apache tomcat server . as shown in figure 2 , different search forms are available through the homepage . the entry point trna allows searching by organism , genome ( nuclear , plastidial and/or mitochondrial ) , amino acid and anticodon and gives access to trna gene lists . species allows access to global information ( including number of trna genes , presence of suppressor or selenocysteine trna genes ) . a summary trna table provides access to trna gene lists and individual biological data . for each trna gene of a trna gene list , this includes organism , chromosome , position , trna type , anticodon , upstream and downstream sequences , intron sequences and mitochondrial import . trna gene sequences and optional information can be downloaded in xls or fasta file formats . although the database is focused on true trna genes ( by means of manual curation ) , we annotated some of the sequences as pseudogenes . we also annotated numerous non - expressed mitochondrial or plastidial trna gene sequences inserted into nuclear genomes and inadequately recognized by trnascan - se as true trna genes . these sequences as well as the sequences of previously identified short interspersed trna - related elements ( 12,24 ) can also be downloaded in xls or fasta file formats . enzymes allows access to a list of aminoacyl - trna synthetases and processing or modification enzymes alongside with their accession numbers , subcellular localization ( experimentally validated or predicted by appropriate organellar targeting prediction programs such as predotar or targetp ( 25,26 ) ) . finally , a blast search entry is available and key references are provided . in total , 3821 , 368 and 186 trna genes were registered from the nuclear , plastidial and mitochondrial genomes , respectively , of the 11 photosynthetic organisms ( note that the mitochondrial dna sequences of p. trichocarpa , m. truncatula and b. distachyon are not available ) . the usefulness of plantrna implementation is illustrated below by a non - exhaustive list of data that can be retrieved from its web interface . first , the selection of intron - containing trna genes shows that all photosynthetic organisms studied here possess trna genes with introns but their number and identity greatly vary ( figure 1 ) . the same two families of nuclear trna genes ( trna gene and elongator trna gene ) contain intronic sequences ( between positions 37 and 38 of their trna sequences ) in flowering plants and in the bryophyte physcomitrella patens , while many more trna genes contain introns in the two green algae . in the two stramenopiles ( diatom and brown alga ) , trna genes corresponding to eight amino acids possess introns in p. tricornutum , while only the family of trna gene contains intron sequences in e. siliculosus , thus demonstrating the independent acquisition of intron sequences among phylogenetically related species . interestingly , it is worth to note that in all photosynthetic organisms , intron sequences are always found in trna gene . this intron acquisition thus very likely occurred before the divergence between plants and metazoans . in human , trna belongs to the very rare human intron - containing trnas ( 27 ) and is essential for the presence of a pseudouridine residue at position 35 of the anticodon ( 28 ) . second , another evolutionary interesting aspect is the presence of a trna among a eukaryotic genome . the presence of selenoproteins is not restricted to the animal kingdom and the occurrence of sec - containing proteins was reported in algae such as chlamydomonas or in diatoms ( 29,30 ) while higher plants lost the ability to synthesize seleno - containing proteins and no trna is present ( figure 1 ) . however , due to its unusual secondary structure , trna is not often retrieved from genome sequences during genome annotation by trnascan - se using default parameters . for example , while selenoproteins are found in stramenopiles ( 3032 ) , no trna gene sequence had yet been annotated either in the diatom p. tricornutum or in the brown alga e. siliculosus nuclear genomes . here , the trna sequences from these two organisms were identified and added to the database , thus confirming the maintenance of a selenocysteine pathway in evolutionary divergent algae and glaucophytes . third , mitochondria from different eukaryotic organisms either have a limited set of trna genes or no trna genes at all . to compensate this deficiency , mitochondrial import of a variable number of nucleus - encoded trnas has been demonstrated in several organisms ( 19,20 ) . the number and identity of mitochondria - imported trnas greatly vary between species and this is especially true in the plant kingdom . here , based on experimental evidence or on the insufficient number of mitochondrial trna genes , the plantrna database provides information on trna mitochondrial import ( figure 1 ) . while the green microalga o. tauri does not apparently need to import nucleus - encoded trnas , the green alga c. reinhardtii imports most of its mitochondrial trnas . annotating the other mitochondrial trna genes revealed that both stramenopiles , the brown alga e. siliculosus and the diatom p. tricornutum , lack essential trna genes ( including trna ) , thus implying the need to import nucleus - encoded trnas . in the glaucophyte c. paradoxa , mitochondrial trna genes are also missing . very interestingly , this is reminiscent of the absence of trna gene in the mitochondrial genome of the protozoan reclinomonas americana . this mitochondrial genome more closely resembles the genome of the bacterial ancestor at the origin of the present - day mitochondria than do any other mitochondrial dna ( 33 ) . for a yet unknown reason , mitochondrial import of nucleus - encoded trna seems to be the most conserved and the easiest trna import event . finally , eukaryotic nuclear trna genes are usually transcribed by rna polymerase iii ( pol iii ) thanks to highly conserved internal promoters , called a and b boxes . however , upstream elements were also found to greatly contribute to transcription efficiencies of many trna genes . this is particularly true in higher plants where highly conserved tata - like elements in the region between 25 and 35 upstream trna gene sequences followed by caa triplets in the 1 to 10 regions are particularly frequent ( 12 ) . analyzing the upstream sequences of nuclear trna gene in all photosynthetic organisms registered in the plantrna database using weblogo ( 34,35 ) has confirmed the existence of these conserved sequence signatures for the trna genes of the five flowering plants ( supplementary figure s1 ) . interestingly and in contrast , tata and caa motifs are considerably less frequent in the glaucophyte c. paradoxa and the green alga o. tauri and are very rare in the other green alga c. reinhardtii , the brown alga e. siliculosus and the bryophyte p. patens . it is to note that in the diatom , p. tricornutum , while there is no obvious conserved caa sequence , an at - rich region is present between 30 and 40 of the upstream trna gene sequences . the lack of conserved motif in the upstream trna gene sequences of several photosynthetic organisms resembles the situation found in animal genomes where the presence of tata and caa motifs does not occur frequently ( 5 ) . it thus suggests that evolutionary divergent pathways for trna gene expression regulation exist in the photosynthetic kingdom . at the other extremity of nuclear trna gene sequences , as shown in supplementary figure s1 , such stretches of t residues can be found downstream of the majority of trna genes . nevertheless , two exceptions do exist . in the green alga c. reinhardtii , as previously observed ( 24 ) , many downstream trna gene sequences lack such a polyt tail because of the presence of polycistronic trnas , a situation usually not found in eukaryotes . quite strikingly , we also show here that in the moss p. patens , only 40% of the trna genes possess a polyt stretch of at least 4 ts within their 25 nt downstream sequences . this observation suggests a peculiar genomic organization of the trna genes in this organism and hints that an alternative trna transcription termination process might be operative in p. patens . first , information on trna - related enzymes , up to now included for 7 out of the 11 photosynthetic organisms will be implemented as soon as appropriate high - quality genomic annotations will be accessible . second , we will continue to upgrade the quality of the web interface and offer new search possibilities . third , as the number of completed nuclear genomes from other photosynthetic organisms is increasing rapidly , the database will be implemented with the new trna gene sequences and their related biological information on a regular basis . from an evolutionary point of view , this must be achieved not only for whole genome sequences of flowering plants such as potato ( 36 ) , grapevine ( 37 ) or apple ( 38 ) but also for lower photosynthetic organisms such as lycophytes , haptophytes or cryptophytes ( 39 ) . from an environmental point of view , it will be interesting to analyze and compare trna gene content and organization of extremophile photosynthetic organisms such as thellungiella salsuginea , a close relative of arabidopsis but highly resistant to abiotic stresses ( 40 ) . finally , a long - term objective will be to enrich the biological information content of the database , e.g. through the implementation of trna gene expression profiles , the description of occurring trfs and 3d structure models of plant trnas . all published data performed with the help of the plantrna database should refer to this article . centre national de la recherche scientifique ; university of strasbourg ; french agence nationale de la recherche ( anr ) [ anr-09-blan-0240 - 01 , anr-11-bsv8 008 01 ] ; french national program investissement davenir ( labex mitocross ) ; university of strasbourg and the french ministre de leducation et de la recherche [ to b.g . and m.m . ] .

plantrna database ( http://plantrna.ibmp.cnrs.fr/ ) compiles transfer rna ( trna ) gene sequences retrieved from fully annotated plant nuclear , plastidial and mitochondrial genomes . the set of annotated trna gene sequences has been manually curated for maximum quality and confidence . the novelty of this database resides in the inclusion of biological information relevant to the function of all the trnas entered in the library . this includes 5- and 3-flanking sequences , a and b box sequences , region of transcription initiation and poly(t ) transcription termination stretches , trna intron sequences , aminoacyl - trna synthetases and enzymes responsible for trna maturation and modification . finally , data on mitochondrial import of nuclear - encoded trnas as well as the bibliome for the respective trnas and trna - binding proteins are also included . the current annotation concerns complete genomes from 11 organisms : five flowering plants ( arabidopsis thaliana , oryza sativa , populus trichocarpa , medicago truncatula and brachypodium distachyon ) , a moss ( physcomitrella patens ) , two green algae ( chlamydomonas reinhardtii and ostreococcus tauri ) , one glaucophyte ( cyanophora paradoxa ) , one brown alga ( ectocarpus siliculosus ) and a pennate diatom ( phaeodactylum tricornutum ) . the database will be regularly updated and implemented with new plant genome annotations so as to provide extensive information on trna biology to the research community .

INTRODUCTION DATABASE CONTENT AND WEB INTERFACE RESULTS AND DISCUSSION FUTURE DIRECTIONS DATABASE ACCESS SUPPLEMENTARY DATA FUNDING

PMC5404168

proximal hamstring tendinopathy ( pht ) is the result of chronic overload caused by repetitive eccentric contraction . surgical treatment becomes an option for patients with chronic symptoms that do not respond to conservative treatment . this report describes a case of a 48-year - old man , an amateur triathlete , with deep gluteal pain in the left hip for 12 months , leading to a decline in sports performance . debridement of the conjoint tendon and its reinsertion associated with semimembranosus tenotomy showed good results and is thus an option for the treatment of this pathology after 12 months of follow - up . this article provides surgeons with a new surgical option for this debilitating condition with clinical and functional improvement after 12 months of follow - up . diseases and injuries of the tendons are among the most common problems encountered in sports and are difficult to treat with a direct impact on the reduction of performance [ 1 , 2 , 3 , 4 ] . the most common chronic lesions in the lower limbs involve the patellar and achilles tendons . limited information exists about chronic proximal hamstring tendon disorders [ 5 , 6 , 7 ] . proximal hamstring tendinopathy ( pht ) is the result of chronic overload caused by repetitive eccentric contraction in hamstrings and in conjoined tendon origin [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ] . it has been seen in athletes of various sports activities , especially in jumpers , sprinters and runners of middle and long distance [ 1 , 9 , 10 , 11 ] . the main finding of pht is chronic pain in the lower gluteal region and progressive pain during sports activity , especially during running with a faster pace or sprinting , and most of them also suffer from pain at the site of the ischial tuberosity while sitting for a prolonged time , occasionally radiating to the midthigh [ 6 , 7 , 9 ] . no acute event is associated with the onset of pain , but most patients report prior hamstring injuries . magnetic resonance imaging ( mri ) is the method of choice for the diagnosis of this condition [ 12 , 13 , 14 ] . the initial treatment is conservative and follows the same principles applied to other tendinopathies , including relative rest and ice to relieve symptoms , nonsteroidal anti - inflammatory drugs ( nsaids ) , physical therapy and intratendinous injections of platelet - rich plasma ( prp ) or corticosteroids [ 14 , 15 ] . however , up to 20% of patients fail to respond to conservative treatment and remain symptomatic for longer than 6 months . surgical treatment becomes an option for patients with chronic , debilitating and refractory symptoms associated with typical imaging findings of pht [ 7 , 11 , 12 ] . this study presents a case of refractory pht - treated surgically through modification of an existing technique for pht treatment . the ethics committee at the hospital madre teresa / brazil approved this study , and a written informed consent was obtained from the patient s family before inclusion in the study . a 48 years , male , triathlete with 1.82 m , 69 kg and body mass index of 20.8 kg / m presented at the orthopedic department of hospital madre teresa / brazil in january 2015 . the patient reported chronic pain in the deep gluteal region with 12 months of evolution , exacerbated mainly by race and while sitting for long periods , which caused a significant drop in his athletic performance after the onset of symptoms . pain had nonradiating with the absence of neurovascular symptoms and without any episode of acute trauma . physical examination demonstrated pain on palpation of the left ischial tuberosity and pain at the origin of the hamstrings caused by passive stretching of these muscles . the three pain provocation tests examined ( puranen orava , bent - knee stretch and modified bent knee stretch tests ) were positive . the victorian institute of sport assessment proximal hamstring tendons ( visa - h ) questionnaire on initial presentation is 23 . the mri revealed thickening of the proximal hamstrings complex with intrasubstancial heterogeneity and rupture , besides bone edema in ischial tuberosity ( fig . 1 ) . the patient underwent conservative treatment for 6 months with relative rest , nsaids , specific physiotherapy and percutaneous corticosteroid injection guided by ultrasound . due to maintenance of pain and functional limitation , magnetic resonance images of a 48-year - old male triathlete with chronic posterior left thigh pain . t2-weighted ( tr / te 3779/100 ) axial ( a and b ) and coronal image ( c and d ) at common hamstring insertion level . thickening of the proximal hamstrings complex with intrasubstancial heterogeneity and rupture , beside bone edema in ischial tuberosity . surgery was performed under spinal anesthesia with the patient in the prone position and the knee slightly flexed to relax the hamstring muscles and the sciatic nerve . a transversal posterior incision in the gluteal fold centered in the ischial tuberosity of 6 cm was made . the lower edge of the gluteus maximus muscle was freed , and the posterior cutaneous femoral nerve was identified and spared . the ischial tuberosity was easily exposed by retraction of the inferior border of the gluteus maximus muscle . 2 ) , and a longitudinal and transversal opening of the conjoint tendon with debridement of degenerated tissue was made . the remnant tendon was then reinserted into the ischial tuberosity with two metal anchors 5.0 mm by krackow suture ( corkscrew , arthrex , fa , usa ) . after this , a transverse tenotomy was done to the thickened semimembranosus tendon 4 cm distal to the origin . the tenotomized semimembranosus tendon was then sutured securely to the biceps femoris tendon to prevent excessive retraction . after tenotomy , the sciatic nerve was explored , and the adhesions were freed ( figs . 3 and 4 ) . anatomy of hamstring origin - the conjoint tendon is formed for long head of the biceps femoris laterally and medially by semitendinosus tendon with an oval shape ( 57.4% of the total area ) . the tendon of the semimembranosus originates on the lateral aspect of the ischium , anterolaterally the conjoint tendon , having a crescent - shaped ( 42.6% of the total area of the proximal hamstring complex ) . the average distance between the lateral border of the ischium to the sciatic nerve is 1.2 cm . radiographic evaluation of hip with two metal anchors 5.0 mm by krackow suture ( corkscrew , arthrex , fa , usa ) in left ischial tuberosity . ( a ) the proximal attachment sites of the hamstring muscles were identified and a longitudinal and transversal opening of the conjoint tendon with debridement of degenerated tissue was made . ( b ) the remnants of conjoint tendon was then reinserted into the ischial tuberosity with the help of two metal anchors and a tenotomy is done to the tendinous part of the semimembranosus muscle 4 cm distal to the origin . ( c ) the distal head of the tenotomized semimembranosus tendon is sutured to the biceps femoris tendon . postoperatively , the patient was immobilized with a brace ( x - act rom hip brace , donjoy , ca , usa ) in hip extension for 3 weeks . the patient was allowed to begin full weight bearing gradually during the first 3 weeks following surgery . isometric and eccentric muscle exercises and bicycling with gradually increasing time and intensity were initiated after 4 weeks . on physical examination , the patient had significantly decreased tenderness to palpation of the proximal hamstring tendon , decreased pain with the aforementioned provocative maneuvers , and a significant decrease in pain with sitting ( maximum , 1/10 intensity ) . he was able to begin speed training approximately 12 weeks after the initial examination , at which point his visa - h score was 83 and he had complete resolution of the previously reported pain complaints with forward bending at the trunk or sitting . he continued to do the same eccentric hamstring exercise on the treadmill without change of form . 1 year after initial evaluation , the patient had not had a recurrence of pain , had a visa - h score of 100 , and had returned to competition 8 months after procedure . surgery was performed under spinal anesthesia with the patient in the prone position and the knee slightly flexed to relax the hamstring muscles and the sciatic nerve . a transversal posterior incision in the gluteal fold centered in the ischial tuberosity of 6 cm was made . the lower edge of the gluteus maximus muscle was freed , and the posterior cutaneous femoral nerve was identified and spared . the ischial tuberosity was easily exposed by retraction of the inferior border of the gluteus maximus muscle . 2 ) , and a longitudinal and transversal opening of the conjoint tendon with debridement of degenerated tissue was made . the remnant tendon was then reinserted into the ischial tuberosity with two metal anchors 5.0 mm by krackow suture ( corkscrew , arthrex , fa , usa ) . after this , a transverse tenotomy was done to the thickened semimembranosus tendon 4 cm distal to the origin . the tenotomized semimembranosus tendon was then sutured securely to the biceps femoris tendon to prevent excessive retraction . after tenotomy , the sciatic nerve was explored , and the adhesions were freed ( figs . 3 and 4 ) . anatomy of hamstring origin - the conjoint tendon is formed for long head of the biceps femoris laterally and medially by semitendinosus tendon with an oval shape ( 57.4% of the total area ) . the tendon of the semimembranosus originates on the lateral aspect of the ischium , anterolaterally the conjoint tendon , having a crescent - shaped ( 42.6% of the total area of the proximal hamstring complex ) . the average distance between the lateral border of the ischium to the sciatic nerve is 1.2 cm . radiographic evaluation of hip with two metal anchors 5.0 mm by krackow suture ( corkscrew , arthrex , fa , usa ) in left ischial tuberosity . ( a ) the proximal attachment sites of the hamstring muscles were identified and a longitudinal and transversal opening of the conjoint tendon with debridement of degenerated tissue was made . ( b ) the remnants of conjoint tendon was then reinserted into the ischial tuberosity with the help of two metal anchors and a tenotomy is done to the tendinous part of the semimembranosus muscle 4 cm distal to the origin . ( c ) the distal head of the tenotomized semimembranosus tendon is sutured to the biceps femoris tendon . postoperatively , the patient was immobilized with a brace ( x - act rom hip brace , donjoy , ca , usa ) in hip extension for 3 weeks . the patient was allowed to begin full weight bearing gradually during the first 3 weeks following surgery . isometric and eccentric muscle exercises and bicycling with gradually increasing time and intensity were initiated after 4 weeks . on physical examination , the patient had significantly decreased tenderness to palpation of the proximal hamstring tendon , decreased pain with the aforementioned provocative maneuvers , and a significant decrease in pain with sitting ( maximum , 1/10 intensity ) . he was able to begin speed training approximately 12 weeks after the initial examination , at which point his visa - h score was 83 and he had complete resolution of the previously reported pain complaints with forward bending at the trunk or sitting . he continued to do the same eccentric hamstring exercise on the treadmill without change of form . 1 year after initial evaluation , the patient had not had a recurrence of pain , had a visa - h score of 100 , and had returned to competition 8 months after procedure . pht was first described by puranenand orava in 1988 with the name of hamstring syndrome [ 8 , 9 ] . the cause and pathophysiology of tendinopathy in humans have not yet been scientifically known but it has been proposed that the tendon s failed healing response to repetitive stretch and mechanical overload may be associated to the development of tendinosis [ 6 , 8 ] . the differential diagnosis should consider acute hamstrings injury , piriformis syndrome , ischial bursitis , and chronic posterior thigh compartment syndrome [ 12 , 17 , 18 ] . the same principles apply to other tendinopathies including relative rest and ice to relieve symptoms , nsaids drugs , eccentric strengthening of the hamstrings , and core stabilization [ 6 , 19 ] . the time required for full recovery ranges from 1 to 3 months , and most patients have good results and return to sport activities , however , up to 20% of patients have conservative treatment failure and remain symptomatic for longer than 6 months [ 7 , 19 , 20 ] . in refractory cases to conventional treatment , other measures can be tried such as infiltration with corticosteroids , prp therapy or shock wave [ 14 , 15 ] . the surgical procedure is an alternative for patients with chronic , debilitating , and refractory symptoms . the semimembranosus tenotomy is the classical choice for being one of the most affected in pht , transferring stress from the semimembranosus tendon to the biceps femoris and to the semitendinosus muscles . this stress - shielding may assist the semimembranosus tendon to recover [ 8 , 12 ] . the difference of the technique presented in this case relates to the fact of incorporating debridement of degenerated tissue with reinsertion of remnant tendon into the ischial tuberosity , which until the present has not been described for the treatment of chronic conditions ; this reinsertion takes into account the anatomy of the proximal hamstring described by philippon et al . . the elimination of fibrous scar tissue combined with intermuscular suturing allows integral healing of muscle to muscle . it is believed that this decreases the chances of re - injury by eliminating the remodeling tissue that attempts to heal the musculotendinous injury . in addition , it allows the muscle to heal to the adjacent muscle in a tension - free manner , and thus , a stronger healing process will occur [ 2 , 11 , 18 ] . the previous studies have shown that satisfactory results can often be expected after surgical treatment of pht , even after failed conservative therapy [ 5 , 8 ] . reported a semimembranosus tenotomy and exploration of the sciatic nerve with 89% of excellent and good results after 49 months of follow - up . performed partial or multiple punctures to relax the myotendinous unit with an excellent result in 88% and good in 12% of cases , respectively . the limitations of this study are the report of a singular case and relatively short follow - up , which means that data must be interpreted with caution when extrapolated ; however , the results are significant due to the rarity of injury and specific treatment . because of low description , most of the published studies are case reports , so comparative studies addressing different methods have not been reported . this article provides surgeons with a new surgical option for this debilitating condition with clinical and functional improvement after 12 months of follow - up . surgical treatment becomes an option for patients with chronic , debilitating and refractory symptoms associated with typical imaging findings . debridement of the conjoint tendon and its reinsertion associated with semimembranosus tenotomy is a new technique described and has shown good results and is thus an option for the treatment of this pathology after 12 months of follow - up .

introduction : proximal hamstring tendinopathy ( pht ) is the result of chronic overload caused by repetitive eccentric contraction . surgical treatment becomes an option for patients with chronic symptoms that do not respond to conservative treatment.case report : this report describes a case of a 48-year - old man , an amateur triathlete , with deep gluteal pain in the left hip for 12 months , leading to a decline in sports performance . magnetic resonance imaging revealed abnormalities that suggested a pht . surgery was indicated following the failure of conservative treatments . debridement of the conjoint tendon and its reinsertion associated with semimembranosus tenotomy showed good results and is thus an option for the treatment of this pathology after 12 months of follow-up.conclusion:this article provides surgeons with a new surgical option for this debilitating condition with clinical and functional improvement after 12 months of follow - up .

Introduction: Case Report: Conclusion: Introduction Case Report Surgical technique Discussion Conclusion

PMC5063014

the rising incidence and severity of clostridium difficile - associated infection ( cdi ) require a rapid and accurate laboratory diagnosis supporting the therapy and prevention of this important nosocomial infection . a two - step approach incorporating the detection of glutamate dehydrogenase ( gdh ) for screening and the detection of the exotoxins tcda and tcdb ( toxins a&b ) as a confirmatory test belongs to the most widely accepted techniques recommended by american and european guidelines . among the several devices available for this two - step diagnostic procedure , we evaluated the performance of the liaison analyzer ( diasorin , italy ) , employing a chemiluminescent immunoassay ( clia ) to identify gdh and the toxins a&b , and compared it to the minividas testing system ( biomrieux , france ) , relying on an enzyme - linked fluorescent immunoassay ( elfa ) to detect toxins a&b and , as a gold standard test , to stool cultures on a c. difficile - selective medium ( clo agar , biomrieux , france ) . within this study , sensitivities , specificities , and positive and negative predictive values were investigated . furthermore , performance under routine conditions was assessed . a total of 249 native fecal specimens were collected from patients with a suspected cdi from september to december 2014 at a tertiary care hospital . the samples were inoculated onto c. difficile selective agar ( clo , biomrieux , france ) and incubated at 37 c for 48 h in an anaerobic pouch ( genbag , biomrieux , france ) . the involved bacterial species from morphologically characteristic colonies was identified via latex agglutination ( c. difficile test kit , oxoid , uk ) . all fecal specimens were tested with the minividas system , employing a one - step fluorescent sandwich immunoassay for the presence of toxins a&b according to the manufacturer s instructions . c. difficile strains isolated from toxin - negative stool samples were analyzed for toxin production with the minividas system as described previously . in parallel , all samples were examined with the liaison platform for the presence of gdh and toxins a&b according to the manufacturer s instruction with the aim to compare its analytical performance to the minividas system . the liaison analyzer uses a two - step chemiluminescent sandwich immunoassay with one monoclonal antibody on paramagnetic beads for capture and polyclonal antibodies for detection of the test molecules . gas - freed millipore water was used to operate the system . c. difficile strains isolated from toxin - negative stool samples were tested for toxin production not only with minividas but also with liaison . sensitivities , specificities , and positive and negative predictive values with 95% confidence intervals were determined . the upper boundary was stated as 100% in the case of calculated values exceeding 100% ( table 1 ) . in our study we compared the technical performance of two ce - marked instruments by running parallel tests from noninvasively sampled material for one parameter specifically ordered by the attending physician . therefore , according to the german medical product law our work did not require ethical approval or consent . a total of 249 native fecal specimens were collected from patients with a suspected cdi from september to december 2014 at a tertiary care hospital . the samples were inoculated onto c. difficile selective agar ( clo , biomrieux , france ) and incubated at 37 c for 48 h in an anaerobic pouch ( genbag , biomrieux , france ) . the involved bacterial species from morphologically characteristic colonies was identified via latex agglutination ( c. difficile test kit , oxoid , uk ) . all fecal specimens were tested with the minividas system , employing a one - step fluorescent sandwich immunoassay for the presence of toxins a&b according to the manufacturer s instructions . c. difficile strains isolated from toxin - negative stool samples were analyzed for toxin production with the minividas system as described previously . in parallel , all samples were examined with the liaison platform for the presence of gdh and toxins a&b according to the manufacturer s instruction with the aim to compare its analytical performance to the minividas system . the liaison analyzer uses a two - step chemiluminescent sandwich immunoassay with one monoclonal antibody on paramagnetic beads for capture and polyclonal antibodies for detection of the test molecules . gas - freed millipore water was used to operate the system . c. difficile strains isolated from toxin - negative stool samples were tested for toxin production not only with minividas but also with liaison . sensitivities , specificities , and positive and negative predictive values with 95% confidence intervals were determined . the upper boundary was stated as 100% in the case of calculated values exceeding 100% ( table 1 ) . in our study we compared the technical performance of two ce - marked instruments by running parallel tests from noninvasively sampled material for one parameter specifically ordered by the attending physician . therefore , according to the german medical product law our work did not require ethical approval or consent . a total of 249 stool samples were examined in 46 analytical runs both with the minividas - elfa c. difficile toxins a&b assay ( vidas cdab ) and with the liaison clia c. difficile gdh and toxins a&b tests in comparison to toxigenic culture as a reference test . sensitivities , specificities , and positive and negative predictive values were determined ( table 1 ) . the gdh - clia assay revealed a high sensitivity ( 98.0% ; ci : 88.299.9 ) and negative predictive value ( 99.5% ; ci : 98.4100 ) . the toxins a&b assays exhibited approximately the same low sensitivity and high specificity , i.e. , 66.7% ( ci : 52.078.9 ) with mini - vidas versus 68.6% ( ci : 54.080.1 ) with liaison , and 99.5% ( ci : 96.8100 ) with minividas versus 100% ( ci : 97.6100 ) with liaison , respectively ( table 1 ) . the two systems did not have a high positive ( 77% ) and negative ( 93% ) concordance since the equivocal test values of the elfa match approximately the same number of positive and negative clia results . detecting toxin production in c. difficile cultures of toxin - negative stool samples via clia and frequent difficulties were experienced when setting up daily controls of the liaison system during the test period ( table 2 ) , leading to repeated error calls . analyses were run on 46 distinct days , and on almost half of them ( 22 days = 48% ) a total of 32 control errors were detected ( table 2 ) . most of the errors ( 56% ) were connected to the light check solution ( table 2 ) . this fluid was used for functionality control of the photomultiplier and for accuracy control of pipetting . the positive controls of the toxins a&b and gdh were linked 22% and 16% of the error messages , respectively ( table 2 ) . on 8 days , double error rates occurred and even a triple error rate was observed on a single day during the testing period . for troubleshooting , all of the control tests had to be rechecked consecutively , resulting in a longer turnover time to the first diagnostic report . repeating the light check added 20 min every time , and repeating the positive controls added 45 min per control to the whole test procedure . a total of 249 stool samples were examined in 46 analytical runs both with the minividas - elfa c. difficile toxins a&b assay ( vidas cdab ) and with the liaison clia c. difficile gdh and toxins a&b tests in comparison to toxigenic culture as a reference test . sensitivities , specificities , and positive and negative predictive values were determined ( table 1 ) . the gdh - clia assay revealed a high sensitivity ( 98.0% ; ci : 88.299.9 ) and negative predictive value ( 99.5% ; ci : 98.4100 ) . the toxins a&b assays exhibited approximately the same low sensitivity and high specificity , i.e. , 66.7% ( ci : 52.078.9 ) with mini - vidas versus 68.6% ( ci : 54.080.1 ) with liaison , and 99.5% ( ci : 96.8100 ) with minividas versus 100% ( ci : 97.6100 ) with liaison , respectively ( table 1 ) . the two systems did not have a high positive ( 77% ) and negative ( 93% ) concordance since the equivocal test values of the elfa match approximately the same number of positive and negative clia results . detecting toxin production in c. difficile cultures of toxin - negative stool samples via clia and frequent difficulties were experienced when setting up daily controls of the liaison system during the test period ( table 2 ) , leading to repeated error calls . analyses were run on 46 distinct days , and on almost half of them ( 22 days = 48% ) a total of 32 control errors were detected ( table 2 ) . most of the errors ( 56% ) were connected to the light check solution ( table 2 ) . this fluid was used for functionality control of the photomultiplier and for accuracy control of pipetting . the positive controls of the toxins a&b and gdh were linked 22% and 16% of the error messages , respectively ( table 2 ) . on 8 days , double error rates occurred and even a triple error rate was observed on a single day during the testing period . for troubleshooting , all of the control tests had to be rechecked consecutively , resulting in a longer turnover time to the first diagnostic report . repeating the light check added 20 min every time , and repeating the positive controls added 45 min per control to the whole test procedure . both delays severely interfered with the daily routine workflow . the liaison analyzer is a popular chemiluminescent platform for testing clinical biochemical and serological parameters with excellent analytical performance and a complex and sensitive operative system . the liaison test kits c. difficile gdh and c. difficile toxins a&b were employed in a two - step approach to diagnose cdi . the gdh - clia test showed a high sensitivity and negative predictive value ; hence , it is well suited as a screening test in the two - step diagnostic approach in cdi in accordance to previous reports [ 6 , 7 ] . the toxins a&b - clia along with the comparator elfa on the minividas device excel with low sensitivity and high specificity in agreement with the previous reports as well [ 4 , 8 ] , indicating that both assays perform well as confirmatory tests . in the case of clia , the specificity was 100% , pointing that the liaison c. difficile toxins a&b test could be the confirmatory test of choice . previous reports about c. difficile diagnostics on the liaison analyzer focused only on the analytical performance but did not evaluate the technical handling of the device . with error messages on 48% of the days when test runs were performed , working with liaison is cumbersome and time - consuming . test errors result in a loss of time by an average of 22 min , therefore , interfering with the daily routine workflow . during the implementation and verification of new diagnostic methods , it is crucial to ensure that drawbacks , due to internal systematic errors , are rapidly and smoothly eliminated through appropriate preparatory training of the personnel . our technicians were thoroughly and specially trained by the manufacturer before starting to work with the liaison analyzer ; therefore , typical sources of errors like , e.g. , the utilization of not properly gas - freed millipore water , could have been excluded . however , dispatch of the above mentioned errors was not possible , causing a constant daily hindrance in the diagnostics of c. difficile - associated infection . the liaison analyzer and the minividas system revealed a comparable analytical performance in the laboratory diagnosis of clostridium difficile - associated enterocolitis . nonetheless , due to numerous technical drawbacks experienced with the liaison analyzer , the time to the first diagnostic report was delayed and the laboratory workflow was consecutively hampered . especially , these two latter issues should individually be considered when thinking about establishing a two - stage diagnostic approach to diagnose clostridium difficile enterocolitis .

backgroundchemiluminescent or enzyme - linked fluorescent immunoassays are commonly used to diagnose clostridium difficile - associated diarrhea.methodsthe liaison analyzer ( diasorin , italy ) was compared to minividas ( biomrieux , france ) and , furthermore , to culture of toxigenic strains . in total , 249 native stool samples were analyzed . sensitivities , specificities , and positive and negative predictive values were investigated . furthermore , performance under routine conditions was assessed.resultsthe glutamate dehydrogenase chemiluminescent immunoassay ( gdh - clia ) assay revealed a high sensitivity and negative predictive value . the toxins a&b assays exhibited approximately the same low sensitivity and high specificity . technical drawbacks experienced with the liaison analyzer in 48% of the analyses considerably delayed the time to the first diagnostic report and interfered with laboratory routine workflow.conclusionthe analytical performance of the investigated platforms should be reflected in the context of implementation into the laboratory workflow .

Introduction Materials and methods Stool samples Stool culture Identification of GDH, TcdA, and TcdB Statistics Ethics Results Analytical performance Technical performance Discussion Conclusion

PMC4733298

a perfect hypospadias repair is supposed to return urethral continuity with sufficient caliber , eradicate phallus curvature , and supply an acceptable appearance with low complications . in this study we are planning to assess objectively this skillful technique for complications such as fistula , dehiscence , recurrent ventral curvature , meatal stenosis , diverticulum and urethral stricture . from june 2012 to december 2013 onlay island flap was performed to repair hypospadias with shallow urethral plate measuring less than 6 millimeter . island flap onlay urethroplasty was performed based on the unique introduction by duckett ( 1 ) . in all cases glans penis is injected with 1:200,000 epinephrine at the incision sites , by using 2.5 magnifying loupe for most cases . ventral skin was incised with a u - shaped incision that preserved the urethral plate ( figure 1 ) . subsequently , an artificial erection was performed to notice remaining ventral curvature , which if present , was straightened by nesbit s dorsal plication with 4.0 prolen at the 12 oclock position . cases with severe chordee that needed transect urethral plate underwent inlay genital graft from scrotal skin and onlay island flap urethroplasty was performed 6 months later . then a rectangular flap 1 cm longer than the length of the urethral plate was harvested from the dorsal inner prepuce ventrally rotated and anastomosed to the urethral plate with a 6-o polydioxanone running suture over an 8 fr silastic foley catheter ( figure 2 ) and a vascular pedicle flap was used to cover the suture line . two lateral incisions were made along the urethral plate with deep dissection into the glanular wings except in cases with very small glans . after glansplasty the penile shaft skin was closed with a midline suture or with a transverse island skin flap in cases of significant ventral skin deficiency according to surgeon s preference ( figure 3 ) . in cases with very small glans , urethroplasty was performed without glansplasty . the dressing was changed on day 2 post surgery and catheter removed on day 7 . children under 2 years of age had a double nappy whereas older children had a collecting bag . patients were visited at the time of catheter removal 2 weeks and then at 1 , 3 , 6 and 12 months postoperative . regular weekly meatal dilatation was used only in patients with voiding difficulty that had an obvious tendency to stenosis and a narrow stream on visual observation . type of hypospadiasis , type of ventral curvature repair , complication rate such as fistula , dehiscence , recurrent ventral curvature , meatal stenosis , diverticulum and urethral stricture , management of complications and number of reoperations were analyzed . data were collected and processed using the commercially available software package ( spss-20 for windows ) . twenty three patients with mean age of 30 ( range 10 - 60 ) months underwent onlay island flap repair ; all had a shallow urethral plate < 6 mm , 3 had a very small glans , and 18 had chordee . meatus was located in distal shaft in 5 cases , mid shaft in 8 , proximal in 6 and penoscrotal type in 4 patients . the flap was sutured completely to the urethral plate , except in the mentioned 3 cases that was left 1 cm distal to the tip of the glans , which was sutured to the edges of the glandular wings . glansplasty was performed in all cases , except in mentioned 3 cases that were left 1 cm distal to the tip of the glans , which was sutured to the edges of the glandular wings . chordee was corrected with nesbit s dorsal plication in 16 cases . in 2 cases with severe chordee , the urethral plate was transected first , then underwent inlay genital graft from scrotal skin and onlay island flap urethroplasty was performed 6 months later . following onlay flap repair 3 ( 13% ) boys had fistula ( one in distal shaft and two in midshaft ) , of whom one case with distal fistula is under observation , while other 2 cases were repaired surgically without further complications . all cases that had glansplasty have acceptable voiding and satisfactory cosmetic esthetic result and appearance , except 3 patients who had no glansplasty . it necessitates the construction of a straight penis , with an acceptable caliber of neourethra that ends in a natural slit - like meatus ( 2 ) . in hypospadias repair , there are many different techniques and modifications . technique of repair is based on a number of variants such as degree of curvature , site of meatus , width of urethral plate and surgeon favorite . in onlay flap repair careful protection of the vasculature of the flap and prevention of overlapping suture lines generate a waterproof closure with minimum risk of postoperative fistula . tubularized incised plate ( tip ) is a common operation in hypospadias reconstruction , but our experience shows that risk of stricture and fistula in this method is relatively high and needs acceptable width of urethral plate for urethroplasty . however in sozubir s et al.s study , complications after tip repair were equivalent to other current repairs , and with caution in technical details these complications could be decreased . the mentioned authors believed that this procedure regularly will generate a vertical meatus , and result in a normal aesthetic outcome ( 3 ) . the main complication in their patients was fistula . despite the use of a dartos flap in all cases , fistula occurred in 5% of distal and 19% of proximal repairs ( 4 ) . they used tubularized incised - plate urethroplasty for hypospadias reoperation but when it was employed in proximal hypospadias , they encountered a complication rate of 33% with 21% incidence of fistula and persistent chordee in some patients ( 4 ) . in our study we had fistula in 3 ( 13% ) boys ( one in distal shaft and two in midshaft ) . a large multicenter series of patients with both distal and proximal hypospadias performed tip repair with less than 1% occurrence of fistulas . they approximated the corpus spongiosum over the neourethra during proximal repair and protected neouretra with dartos layer and glans wings . in recent study the only parameter for selection of patients was urethral plate diameter less than 6 millimeter and type of hypospadias was not an effective factor . ( 6 ) in a single - center experience with 500 cases reported tip procedure as a reliable technique for management of both distal and proximal hypospadias in both primary and re - operative cases with a small rate of complications but urethral plate diameter was not mentioned . postoperative meatal / neourethral stenosis after tip is common , so shimotakahara ( 7 ) collected a dorsal inlay graft from the inner prepuce and sutured to midline incision of the urethral plate . in our study 2 cases with severe chordee needed to transect urethral plate who underwent inlay genital graft from scrotal skin and onlay island flap urethroplasty was performed 6 months later and both cases had acceptable results . although tip urethroplasty is a choice procedure in distal penile hypospadias for some surgeons , some others prefer to use onlay flap technique , particularly in cases of a small phallus with narrow plate or conical glans , which create tubularization difficult ( 7 ) . in 1987 , elder ( 8) reported the first one - stage hypospadias repair using an onlay island flap , although the preputial island flap had long been done previously . ( 9 ) evaluated the consequences of using a distally folded onlay flap in the repair of distal penile hypospadias in 36 patients , they had only two urethrocutaneous fistulas , and they used onlay flap for distal type . in our study ( 10 ) had a studied forty five boys with similar mid - penile hypospadias deformities and designed a comparative study between tip and onlay preputial island flap and found no differences between the two techniques . leslie et al . ( 12 ) used tunica vaginalis graft plus onlay preputial island flap in urethral reconstructive surgery in rabbits in one - stage for complex hypospadias with divided urethral plate . ( 13 ) compared three different urethroplasty techniques ( onlay , buccal mucosa , koyanagi type i ) in severe hypospadias . fistula occurred in 15% in onlay group ; 32% in buccal mucosa group , and 19.2% in koyanagi cases . patel et al . ( 14 ) introduced a technique called the split onlay skin flap , which had fistula in 6 patients . snodgrass ( 16 ) reported most of urethral complications are diagnosed at the rst postoperative visit or within the rst year after reconstruction . glans dehiscence is usually apparent by 2 months , whereas most stulas and other complications are found after 6 months . in our experience with 23 patients the onlay flap hypospadias repair provided excellent cosmetic and functional results . the overall complication rate as well as the rate of postoperative urethrocutaneous fistula was minimal and compares favorably with rates reported by other authors , but need more long postoperative surveillance .

background : hypospadias is one of the most common congenital genital anomalies in males that necessitates to be operated early in infancy ( when 6 to 9 months old ) . on the other hand , hypospadias is a challenging field of pediatric urology with multiple reconstruction techniques . a perfect hypospadias repair is supposed to return urethral continuity with sufficient caliber , eradicate phallus curvature , and supply an acceptable appearance with low complications.objectives:this study aimed to evaluate the outcomes of using onlay island flap technique in the repair of hypospadias with shallow urethral plate.patients and methods : in this prospective study within june 2012 to december 2013 , we performed onlay island flap procedure to repair hypospadias with shallow urethral plate measuring less than 6 millimeter . this technique was selected for all types of hypospadiasis except subcoronal type . nesbit s dorsal plication procedure was established for chordee . in cases with very small glans , urethroplasty was performed without glansplasty.results:twenty three patients with mean age of 30 ( range 10 - 60 ) months underwent onlay island flap repair ; all had a shallow urethral plate < 6 mm , 3 had a very small glans , and 18 had chordee . meatus was located in distal shaft in 5 cases , mid shaft in 8 , proximal in 6 and penoscrotal type in 4 patients . chordee was corrected with nesbit s dorsal plication in 16 cases . complications were : meatal stenosis in 2 cases and urethrocutaneous fistula in 2 patients , all of which were repaired surgically . mean follow up time was 13 ( 3 - 20 ) months . all cases that had glansplasty have excellent esthetic appearance.conclusions:this technique offers acceptable results regarding meatal stenosis , urethrocutaneous fistula and esthetic outcome .

1. Background 2. Objectives 3. Patients and Methods 4. Results 5. Discussion 5. 1. Conclusion

PMC3567317

physical inactivity is a leading cause of most non - infectious diseases [ 1 - 3 ] , including heart disease , some cancers , and type 2 diabetes . non - infectious diseases account for approximately 60% of the 56 million deaths around the world annually and 47% of the global burden of disease , and this burden continues to grow . these statistics demonstrate the importance of physical activity for healthy living and illustrate that physical inactivity and the associated health - related problems have no geographical boundaries . in light of this , commitment to sustained physical activity across the lifespan is imperative for personal health , and is a primary goal of physical activity specialists around the world . when striving to promote the commitment to sustained physical activity , physical activity specialists are most effective when utilizing the best available research to guide their practice . at the heart of sound research is theory , making sound theory a key to effective practice . to be sound , a theory must be both practical and parsimonious . this leads to the question , " what is the best theory to aid physical activity specialists in promoting a commitment to life - long physical activity ? " the purpose of this paper is to review the literature in search of a practical and parsimonious theory that applies to and explains commitment to both sport and exercise for children , adolescents , and adults . the sport commitment model is an evolving theory that explains participation in physical activity in the sport context . physical activity is commonly defined as any bodily movement produced by skeletal muscles that results in energy expenditure , including sport and exercise . sport is a form of physical activity that is highly organized and socially competitive , while exercise is physical activity that is planned , structured , repetitive , and designed to improve or maintain physical fitness . for the specific purposes of this paper , the term physical activity refers to sport and exercise collectively because , as argued in the paper , sport and exercise motivation from a commitment perspective are more similar than different and one unified model is sufficient and parsimonious . however , to maintain the integrity of the original research discussed in this paper , original terms used by authors ( e.g. , sport or exercise ) are used in this paper . motivation for participation is manifested in individuals ' behavior in the form of their activity choices , how much effort they put forth in physical activity - related endeavors , and how long they persist , particularly in the face of obstacles and failure . of particular interest to physical activity and health professionals is creating environments in which individuals not only participate in physical activity , they will persist in being physically active . research shows that regardless of age , youth and adults participate in physical activity for similar reasons . when asked why they participate , youth sport participants responses generally fall among the following categories : fun , competence , affiliation , and fitness . that is , people , young and old , participate in sport and exercise , because it is fun , they like to strive to achieve and experience feelings of competence , they want to be with their friends and meet new people , and they see physical activity as an avenue for increasing fitness . despite the many perceived benefits of participation , people also withdraw from physical activity . in the united states , approximately one - third of all participants in organized youth sports drop out yearly with the estimated average youth sport attrition rates ranging from 22% to 59% . there has been a decrease in participation among popular sport and fitness activities including , tennis , racquetball , aerobic dance , jogging / running , and weight lifting , and it is commonly reported that 50% of adults who begin an exercise program drop out within 3 to 6 months . to better understand sport participants ' motivation , scanlan et al . , scanlan et al . these researchers defined sport commitment as a motivational force that reflects a person 's desire and resolve to continue participation in sport . sport commitment is a dynamic , psychological state that can vary over time , through seasons , and over the course of careers . individuals ' level of commitment influences behavior in the form of choice , persistence , and effort . through a combination of qualitative and quantitative studies , scanlan et al . identified six determinants of sport commitment including sport enjoyment , involvement opportunities / benefits , personal investments , social support , involvement or attractive alternatives , and social constraints . personal investments are the personal resources such as time , energy , money , and effort that are put into the activity that can not be recovered if participation is discontinued . social support is the participant 's perceived support and encouragement from significant others in response to the participant 's involvement in sport . involvement alternatives refer to the attractiveness or allure of other activities relative to one 's current sport choice . social constraints reflect expectations that create feelings of obligation to others such as parents , peers , and coaches to continue involvement in sport . of the six determinants , enjoyment , involvement opportunities , personal investments and social support have been found to be positively related to sport commitment among youth , elite - amateur , and professional sport athletes [ 13 - 17 ] . of these , though they have been found to predict commitment , involvement alternatives and social constraints have not consistently predicted commitment across all groups . for example , involvement alternatives has been shown to significantly predict sport commitment in adult and high school athletes . however , younger athletes do not appear to have the same understanding of this construct , possibly because younger athletes may not have to make competing choices regarding sport . it is interesting to note that although social constraints is theorized to be positively related to sport commitment , research has demonstrated small negative or no relationship between social constraints and sport commitment in adult or youth sport [ 13 - 18 ] . these individuals may be participating because of their personal desire rather than feelings of obligation . that is , participants play because they want to , not because they have to . the concept of individuals playing because they want to reflects an attraction - based commitment , which some researchers refer to as " want to " commitment . conversely , the notion that individuals play because they have to play , they have no choice , reflects an entrapment - based form of commitment or " have to " commitment . also , individuals are theorized to play with low commitment levels . though not addressed by early sport commitment researchers , these three types of commitment were part of the early conceptualization of the sport commitment model by schmidt and stein and can be thought of as profiles denoting attraction - based ( want to ) , entrapment - based ( have to ) , and low commitment athletes . theoretically , the profiles of attraction - based , entrapment based , and low commitment are distinct ( figure 1a ) . specifically , an attraction - based profile is theorized to reflect high enjoyment , involvement opportunities ( in the form of high benefits and low costs ) , high personal investment , and low involvement alternatives . athletes with an attraction profile are the most likely to continue their sport experience with positive feelings and attitudes . the entrapment - based and low commitment profiles are theorized as distinct , but similar . both profiles are expected to be associated with low enjoyment , low involvement opportunities ( in the form of high costs and low benefits ) , and high personal investment . athletes with low commitment are theorized to see other activities as attractive , whereas entrapped athletes see none . in essence , athletes fitting the entrapment - based profile are motivated to continue playing because they have nothing better to do and/or they have put too much into the sport to quit . in contrast , athletes fitting the low commitment profile do see involvement alternatives , but have put much into the sport . while they may be tempted to quit , they tend to continue participating . specifically , raedeke and weiss and weiss , examined the relationship between commitment profiles and motivation - related behavior . among the findings of these studies , only two profiles were found to be conceptually similar to the theoretical profiles ( figure 1b ) . specifically , raedeke found profiles resembling attraction - based and low commitment among 13 to 18 years old competitive age - group swimmers and weiss and weiss found similar profiles among 10 to 18 years old competitive club gymnasts . interestingly , in neither study did an entrapment - based commitment profile emerge . in both studies , and as expected , the profile resembling the attraction - based profile was more positively associated with motivation . specifically , swimmers in the attraction - based profile reported the lowest burnout , whereas those in the low commitment profile reported the greatest level of burnout . those in the attraction - based profile reported greater intrinsic motivation , effort , and persistent , as well as lower amotivation than those in the low commitment profile . based on these results and theory , coaches who create environments in which their athletes enjoy , value , and invest are likely to have committed athletes . the perceived utility of the sport commitment model has led some to investigate its veracity in exercise- and leisure - related behavior . using the sport commitment model as a guiding framework , wilson and colleagues investigated commitment to exercise . acknowledging that many exercise out of feelings of obligation as well as choice , wilson et al . conceptualized exercise commitment to reflect both functional ( want to ) and obligatory ( have to ) resolve to continue exercising . relative to the sport commitment model , they reconceptualized sport enjoyment as the degree to which exercisers perceive exercise as rewarding and labeled this as satisfaction . all remaining determinants are conceptually similar to the sport commitment model , with the context being shifted from sport to exercise . in their examination of the relationship between exercise commitment and exercise - related behavior , wilson et al . hypothesized : 1 ) two types of commitment including a volitional , attraction - based ( i.e. , want to ) form of commitment and an obligatory , entrapment - based ( i.e. , have to ) form of commitment ; 2 ) exercise commitment would be positively related to satisfaction , personal investment , involvement opportunities and social support , and differentially to involvement alternatives and social constraints ; and 3 ) exercise commitment would be positively related to more frequent and intense exercise behavior . interestingly , no specific hypotheses were made regarding the relationship between the determinants of exercise commitment and " want to " vs. " have to " commitment . partial support was found for their hypotheses based on the responses from 428 university students and staffers enrolled in group exercise classes who completed questionnaires assessing exercise commitment , determinants to exercise commitment , and the frequency in which they engaged in varying levels of exercise . relative to the first hypothesis and using structural equation modeling , they found that exercise commitment was multidimensional with " want to " and " have to " commitment . relative to the second hypothesis involving the relationship between the determinants and commitment , only satisfaction and personal investment positively predicted both " want to " and " have to " commitment , with involvement opportunities and social constraints positively predicting " have to " commitment . interestingly though , and relative to the third hypothesis , only " want to " commitment was related to self - report exercise behavior . these findings reveal that only satisfaction and personal investment relate to exercise behavior through exercise commitment . practically speaking , individuals who experience greater satisfaction exercising and who perceive that they have put a greater investment of resources into exercising ( e.g. , time , money , energy , and effort ) are 1 ) committed to exercise volitionally ( i.e. , because they want to ) and 2 ) more apt to exercise than those who are less satisfied and perceive putting a lower investment of resources into exercise . as with the research on sport commitment , " want to " ( aka , attraction - based ) commitment served as a strong predictor of persistent exercise participation while the concept of " have to " ( aka , obligatory or entrapped ) commitment did not emerge as a predictor of exercise behavior . this review of the sport commitment research has revealed that enjoyment / satisfaction and personal investment are the strongest most consistent determinants of commitment to physical activity . based on this , physical activity specialists are advised to create environments in which people find enjoyment and are willing to invest . while practical and parsimonious , more research is needed to determine the adequacy of this conclusion . can commitment to physical activity be reduced to two determinants and still explain motivation adequately ? when attempting to determine the best and most parsimonious model for fostering a commitment to physical activity through sport and exercise , the investment model should be considered . the investment model , the model from which the sport commitment model emerged , was proposed by rusbult to " predict ( the ) degree of commitment to and satisfaction with a variety of forms of ongoing associations ( e.g. , romantic relationships , friendship , and business ) with wide ranges of duration and involvement " ( p. 173 ) . the similarities between the investment model and the sport / exercise commitment model are evident in the determinants and nature of commitment proposed in both models . rusbult identified five determinants of commitment including satisfaction / attraction to an association , attractive alternatives , investments , the rewards or benefits of an association , and the costs of an association . rusbult 's five determinants of commitment are conceptually consistent with sport enjoyment / exercise satisfaction , involvement alternatives , personal investments , and involvement opportunities , respectively . it is noteworthy that while cost was initially considered by schmidt and stein , it is not a specific determinant of the sport commitment model . second , rusbult acknowledged the multidimensional nature of commitment when she stated that " high investments and/or poor alternatives may sometimes serve to trap the individual in an unhappy , unsatisfying relationship rendering it possible to have high commitment while satisfaction and attraction to the ongoing association are low " ( p. 175 ) . in light of 1 ) the similarities between the commitment models developed in physical activity contexts ( sport and exercise ) and the investment model , 2 ) the original intention for the investment model to predict commitment in a variety of contexts , 3 ) the role of satisfaction and personal investment in explaining physical activity commitment , 4 ) the meaningfulness of the concept of costs in the study of physical activity commitment , and 5 ) the value of conceptual parsimony , the investment model deserves further consideration as a more efficient and equally as effective model for predicting physical activity behavior than the sport commitment model . recently , the investment model was , in part , applied to the exercise setting . examined the relationships among exercise commitment , determinants of exercise commitment , and exercise - related behavior . they assessed both " want to " and " have to " commitment , and exercise behavior was assessed in terms of the participants ' stage of behavior change and time spent in leisure physical activity across seven days . although they did not forward specific hypotheses , it is reasonable to suggest that they expected that 1 ) satisfaction and personal investments would relate positively to both forms of commitment , 2 ) involvement alternatives would relate positively to " want to " and negatively to " have to " commitment , and 3 ) " want to " and " have to " forms of commitment would both relate positively to stages of behavior change and time spent exercising . the responses from 267 individuals participating in university classes , private health clubs , and a community running club were analyzed using structural equation modeling and multivariate analyses . results revealed that satisfaction and personal investments related positively to " want to " and " have to " commitment , but attractive alternatives only related to " have to " commitment . additionally , only " want to " commitment related to exercise - related behavior . collectively , results of these studies support the conclusion that satisfaction and personal investments are two keys to fostering " want to " commitment , and exercise behavior . a review of the literature across sport and exercise in conjunction with the sport commitment and investment models reveals that satisfaction / enjoyment and personal investments are consistent predictors of commitment to persistent participation in physical activity in the form of sport and exercise . additionally , attraction - based or " want to " commitment is a consistent predictor of both sport and exercise behavior . in fact , given the results of the research on sport and exercise commitment , it is can be argued that the investment model may be as practical as and provide a more parsimonious explanation of commitment to sport and exercise than the sport commitment model . more research is needed to clarify the utility and parsimony of the investment model and sport commitment model when promoting long - term participation in sport and exercise across the lifespan , and to extend this literature to include other physical activity settings such as rehabilitation . research employing structural equation modeling and experimental design would contribute greatly to our understanding of commitment to physical activity and finding the most parsimonious explanation to what leads to persistent involvement in physical activity . from a practical standpoint , the research on exercise and sport commitment emphasizes the importance of fostering " want to " physical activity behavior , and affirms that creating fun environments and valued experiences are important factors . first , enjoyment / satisfaction have been consistent predictors of physical activity participation [ 13 - 15 ] . second , some have argued that enjoyment mediates the relationship between commitment and its determinants . lastly , fun is a primary reason people give for participating in physical activity . no doubt examined sources of sport enjoyment . among youth they found that social interactions with coaches , parents , and teammates ; demonstrating one 's ability ; mastering skills ; and being effortful were all sources of fun . when reflecting back on their sport experiences , former elite figure skaters revealed that they found enjoyment in 1 ) the act of skating , 2 ) competitive achievements including skill mastery and demonstrating athletic ability , and 3 ) social opportunities , such as recognition for their accomplishments and ability , friendship opportunities , and relationships with their coaches . when viewed together , sources of enjoyment mentioned by youth and adults are similar . sport is enjoyable and fun , because sport provides opportunities for social affiliation , achievement / mastery , and movement ( fitness ) . these findings regarding sources of enjoyment are noteworthy , particularly in relation to individuals ' motives for participation in physical activity . consider the similarities between the primary reasons youth and adults give for participating in physical activity and their sources of enjoyment . both participate in physical activity for fun and social affiliation , to build competence / strive for success , and for fitness . they enjoy the sport experience through social affiliation , achievement , and movement / play . put within the discussion of sport commitment , if the physical activity experience can be designed to meet people 's motives for participation in the form of social , achievement , and movement opportunities , they will be more likely to enjoy themselves , commit to sport volitionally , and thus be more physically active . in conclusion , the increasing health cost of physical inactivity around the world should impress upon all of us the importance of one 's commitment to physical activity whether through sport or exercise . while the sport / exercise commitment model has provided insight into the understanding of sport commitment , the results highlight the potential relevance and more parsimonious approach of the investment model . the investment model when applied to sport and exercise context may provide an explanation of commitment to physical activity that is as adequate as , and more parsimonious than , the sport / exercise commitment model . no doubt , continued research on commitment to physical activity using and testing both the sport / exercise commitment model and the investment model is necessary to provide a clear understanding of this construct and its relationship with physical activity behavior . collectively , research on sport and exercise commitment to date sends a clear message to physical activity leaders , practitioners , and others desiring to affect physical activity levels and persistence . to foster attraction - based , " want to " commitment to physical activity in the form of sport and exercise , those who enjoy and invest in physical activity are most likely to be committed to participation because they " want to " be involved . it is reasonable to suggest that physical activity professionals can foster a greater attraction - based , " want to " commitment among the youth and adults by focusing on creating fun physical activity environments in which people experience competence , social affiliation , and increased fitness . these environments are more likely to foster greater enjoyment and feelings of personal investment and promote " want to " participation in physical activity .

a commitment to physical activity is necessary for personal health , and is a primary goal of physical activity practitioners . effective practitioners rely on theory and research as a guide to best practices . thus , sound theory , which is both practical and parsimonious , is a key to effective practice . the purpose of this paper is to review the literature in search of such a theory - one that applies to and explains commitment to physical activity in the form of sport and exercise for youths and adults . the sport commitment model has been commonly used to study commitment to sport and has more recently been applied to the exercise context . in this paper , research using the sport commitment model is reviewed relative to its utility in both the sport and exercise contexts . through this process , the relevance of the investment model for study of physical activity commitment emerged , and a more parsimonious framework for studying of commitment to physical activity is suggested . lastly , links between the models of commitment and individuals ' participation motives in physical activity are suggested and practical implications forwarded .

INTRODUCTION PARTICIPATION MOTIVATION THE SPORT COMMITMENT MODEL ATTRACTION, ENTRAPMENT, AND LOW COMMITMENT SPORT COMMITMENT MODEL AND EXERCISE SPORT COMMITMENT MODEL: IS THERE A MORE PARSIMONIOUS EXPLANATION? THE INVESTMENT MODEL APPLIED TO EXERCISE CONCLUSIONS AND PRACTICAL IMPLICATIONS

PMC5400438

vascular graft salvage is successfully manageable with a muscle flap , following wound debridement and targeted antimicrobial therapy , in 7884% of groin infections [ 13 ] . in this context , four types of flaps have been described , including transposition of the sartorius muscle [ 4 , 5 ] , the gracilis muscle [ 5 , 6 ] , the rectus abdominis and rectus femoris muscle . in complicated cases these flaps may , however , not be feasible because of previous use or a compromised vascular supply as their pedicle is distally from the groin . we report a case in which the external oblique muscle was used to salvage an infected vascular graft . an 81-year - old woman , who had been given an aortobifemoral dacron prosthesis in 2007 for occlusive aortoiliac disease , was seen at our clinic with a persisting medial malleolar wound in september 2014 . thrombosis of the left iliac part of the aorta - bifemoral graft explained the insufficient healing . an unsuccessful attempt to recanalize the graft percutaneously required a reconstruction with a femoro - femoral crossover bypass in october 2014 . she was readmitted after blood loss had occurred from her right groin , 6 weeks after the crossover operation . because of the risk of exsanguination due to anastomotic rupture , an emergency exploration was performed and showed incorporation of the largest part of the graft with surrounding tissue . an infected hematoma in contact with the graft was found at the anterior side of her right quadriceps muscle . the sartorius muscle was proximally released and the proximal part of the graft was covered . subcutaneous tissue was closed with a running absorbable suture ( polysorb 3 - 0 , covidien , mansfield , usa ) and the skin was left open ( fig . vacuum - assisted closure ( vac ) ( kci medical , san antonio , usa ) therapy was started immediately postoperatively . bacterial culture confirmed infection with aerobic- and anaerobic bacteria ( proteus vulgaris , citrobacter koseri and bacteroides fragilis ) . in line with the sensitivity of these microorganisms , treatment was started with piperacilline / tazobactam . duplex ultrasound examination 1 week later showed a patent bypass and computed tomography 10 days thereafter did not show any fluid surrounding the graft . white blood cell count remained normal , and c - reactive protein ( crp ) dropped from 111 mg / l to 52 mg / l after 2 months . the hematoma did not seem macroscopically infected ; it was drained through the previous incision and through a new incision laterally , thus diverting the infection away from the graft . anticoagulation for atrial fibrillation was temporarily interrupted , but the wound did not heal well and crp rose to 224 a third exploration was performed 3 days later and again the remainder of a hematoma was removed . a part of the graft was not covered by the sartorius muscle . in a second attempt to cover the graft , the external oblique muscle was released medially from the linea alba and rotated laterally and caudally , into the groin ( fig . 2 ) . figure 2:drawings before ( a ) and after ( b ) reconstruction with sartorius- and oblique external flap . drawings before ( a ) and after ( b ) reconstruction with sartorius- and oblique external flap . granulating tissue showed better vascularization 3 days thereafter , and again crp dropped to 42 mg / l ( fig . bacterial cultures showed the presence of enterococcus faecalis bacteria and antibiotics were changed to amoxicillin with clavulanic acid , combined with ciprofloxacin and was continued for 6 weeks . figure 3:appearance of the wound after removal of negative pressure , 4 days after the oblique external flap . appearance of the wound after removal of negative pressure , 4 days after the oblique external flap . the patient was discharged home 6 days postoperatively and vac - therapy was continued for 2 months , partially at home . figure 4:approximately half a year post oblique flap ; the wound is fully healed without a fistula . approximately half a year post oblique flap ; the wound is fully healed without a fistula . this report indicates that in complicated cases coverage of an infected vascular graft in the groin with the external oblique muscle flap can help preserve the graft and assist in wound closure . the external oblique muscle is a type v muscle with both dominant and multiple segmental vascular pedicles . the dominant pedicles are one or two branches of either the deep circumflex iliac artery ( 94.7% ) or the iliolumbar artery ( 5.3% ) . the use of the external oblique muscle has predominantly been described for chest wall reconstruction . advantages that the external oblique muscle flap offers are its simple release and its lateral vascularization , facilitating rotation . even though it is a very thin muscle , it seems to have the ability to grow in an infected environment to form a large bulky protection of the synthetic bypasses . the vascularization of the external oblique muscle and the non - smoking status of the patient contributed to the favorable outcome . because of the extension of the external oblique muscle cranially , further mobilization is possible , thus enabling coverage of larger areas in the infected groin . one other advantage may be derived from its cranial position and its blood supply ; the viability of the flap does not depend on the vascularization through a ( infected ) bypass graft , as is the case in sartorius- , rectus femoris- and gracilis flaps . also , its blood supply is not dependent on the epigastric artery , essential for rectus femoris flaps . this epigastric / mammary artery is often used in coronary artery bypass surgery ( cabg ) in patients with peripheral artery disease . to the best of our knowledge , external oblique muscle coverage of a vascular graft has not been described previously and is a feasible , effective , technically simple and safe procedure . it is an attractive option in complicated cases , in which previous muscle transpositions were unsuccessful . it can cover large vascular grafts in the groin , particular in patients with poor vascularization distally and in post - cabg patients . a larger series including functional long - term outcomes are required to confirm the above . none of the authors has a financial interest in any of the products , devices or drugs mentioned in this manuscript .

abstractabdominal muscles , such as the oblique- and transverse muscles , find their blood supply from multiple segmental pedicles from the iliac artery . besides its superior vascularization , its release is simple , leaving two abdominal muscles for securing abdominal wall strength . the release of the muscle and coverage of the graft requires partial muscle mobilization and is a minor reconstruction , but extension of the mobilization cranially enables coverage of larger defects . we present a case of an infected vascular graft in the groin successfully preserved through coverage with an external oblique muscle flap .

INTRODUCTION CASE REPORT DISCUSSION FUNDING

PMC4894040

over recent years , laparoscopic surgeries have already been demonstrated as a better alternative to open abdominal surgery , especially when treating benign diseases . intraoperative complications , pain , and length of postoperative hospital stay have all been found to be reduced . many attempts have been made to minimize the number of trocars by applying single port laparoscopic surgery ( spls ) to perform a cholecystectomy , appendectomy , and splenectomy . spls has also been applied to perform hysterectomies by several gynecologic groups with improving effects . however , few studies have evaluated the potential benefits of spls for the treatment of benign adnexal masses ; therefore , in this study , our objective was to assess the feasible advantages of spls via a comparative analysis between spls and conventional laparoscopy for adnexal masses . this study was a case - control study that compared the outcomes of 99 cases of spls and 104 conventional laparoscopic adnexal procedures for adnexal masses between december 2013 and march 2015 performed by a single surgeon . the 99 patients who were chosen to undergo spls were those with the diagnosis of benign adnexal mass . the 104 patients in the control group who underwent conventional laparoscopic adnexal surgery , were age - matched and chosen during the same period . the inclusion criterion for both groups was as follow : patients who were diagnosed with the presence of adnexal masses on ultrasonography and without any severe complications . patients who were under suspicion of malignant ovarian tumor on ultrasonography or with severe complications that can not withstand surgery were excluded . the patients clinical data for both groups including history , routine preoperative workup , such as blood routine , biochemistry , coagulation tests , tumor markers , electrocardiogram , and chest x - ray were all collected retrospectively . all surgeries were performed by a single surgeon ( ling yin ) , with the assistance of two surgeons ( yan zhang and bing - bing xiao ) , at a single institution ( peking university first hospital ) . the postoperative assessment was performed by a single investigator ( si - yun wang ) . the spls port with three access ports ( one 12-mm port and two 5-mm ports ) as well as a gas inlet , which was produced in tonglu , hangzhou , china , was used . after surgeries , intraoperative data including the operation time ( from skin incision to closure ) , estimated blood loss ( ebl ) ( by visual and experiential estimation ) , change in hemoglobin level after surgery ( recorded at 3 days postoperatively ) , duration of postoperative hospital stay , intraoperative and postoperative complications were all collected until 1 month follow - up postoperatively . after a 23 cm transverse transumbilical incision was made , an spls port wound retractor ( 30 mm ) , which was composed of a distal ring , a proximal ring , and a cylindrical connecting sleeve , was then inserted to expand the edge of the incision laterally . the spls port has three access ports ( one 12-mm port and two 5-mm ports ) as well as a gas inlet . we used a rigid , 30 , 10-mm laparoscope , traditional rigid straight instruments , and prebent laparoscopic instruments to prevent the instruments from clashing and to optimize the operation space [ figure 1 ] . ( b ) the spls port has one 12-mm port and two 5-mm ports as well as a gas inlet . ( c ) using a rigid , 30 , 10-mm laparoscope , traditional rigid straight instruments , and prebent laparoscopic instruments to optimize the operation space . once all equipment were in position , the following procedures were similar to the techniques in conventional laparoscopic approach [ figure 2 ] . the skin was closed using a 3 - 0 vicryl suture ( johnson & johnson , usa ) , which provided for a good cosmetic outcome [ figure 3 ] . intraoperative view of a single port laparoscopic surgery for the treatment of an adnexal mass . ( d ) postoperative image . a representive image of the 23 cm incision for the single three - channel port system prior to the final skin closure . the spss version 20.0 ( spss inc . , chicago , il , usa ) was used for statistical analysis using the student 's t - test , fisher 's exact test , chi - square test , and wilcoxon rank sum test . quantitative data that satisfy a normal distribution were analyzed using student 's t - test ( data are shown as mean standard deviation [ sd ] ) . quantitative data that did not satisfy a normal distribution were analyzed using wilcoxon rank sum test ( data are shown as median [ min , max ] ) . qualitative data were analyzed using fisher 's exact test and chi - square test ( frequency [ % ] ) . a value of p < 0.05 was considered as statistically significant . after a 23 cm transverse transumbilical incision was made , an spls port wound retractor ( 30 mm ) , which was composed of a distal ring , a proximal ring , and a cylindrical connecting sleeve , was then inserted to expand the edge of the incision laterally . the spls port has three access ports ( one 12-mm port and two 5-mm ports ) as well as a gas inlet . we used a rigid , 30 , 10-mm laparoscope , traditional rigid straight instruments , and prebent laparoscopic instruments to prevent the instruments from clashing and to optimize the operation space [ figure 1 ] . ( b ) the spls port has one 12-mm port and two 5-mm ports as well as a gas inlet . ( c ) using a rigid , 30 , 10-mm laparoscope , traditional rigid straight instruments , and prebent laparoscopic instruments to optimize the operation space . once all equipment were in position , the following procedures were similar to the techniques in conventional laparoscopic approach [ figure 2 ] . the skin was closed using a 3 - 0 vicryl suture ( johnson & johnson , usa ) , which provided for a good cosmetic outcome [ figure 3 ] . intraoperative view of a single port laparoscopic surgery for the treatment of an adnexal mass . ( d ) postoperative image . a representive image of the 23 cm incision for the single three - channel port system prior to the final skin closure . the spss version 20.0 ( spss inc . , chicago , il , usa ) was used for statistical analysis using the student 's t - test , fisher 's exact test , chi - square test , and wilcoxon rank sum test . quantitative data that satisfy a normal distribution were analyzed using student 's t - test ( data are shown as mean standard deviation [ sd ] ) . quantitative data that did not satisfy a normal distribution were analyzed using wilcoxon rank sum test ( data are shown as median [ min , max ] ) . qualitative data were analyzed using fisher 's exact test and chi - square test ( frequency [ % ] ) . a value of p < 0.05 was considered as statistically significant . the clinical characteristics between the two groups did not differ significantly , including the size of adnexal masses showed on ultrasonography and the proportion of unilateral masses [ table 1 ] . a cystectomy with or without concomitant surgeries such as myomectomy or hysteroscopy was performed in 76.7% of the spls group and in 77.9% of the conventional group , which was not a remarkable statistical discrepancy . the adhesiolysis ratio also showed no obvious difference between the two groups ( spls : 65.7% ; conventional : 67.3% ; p = 0.803 ) ; however , the pathological findings between the two groups were slightly different . in the spls group , mature cystic teratoma made up the vast majority of pathological types while endometrioma was more commonly observed in the control group [ table 2 ] . comparison of preoperative characteristics of the single port laparoscopic surgery group and the conventional group * the size of ovarian tumors was measured by transvaginal ultrasonography ; u value ; t value ; value ; spls : single port laparoscopic surgery ; bmi : body mass index ; sd : standard deviation ; : no data . comparison of operative characteristics of the single port laparoscopic surgery group and the conventional group , n ( % ) * other procedures included myomectomy , hysteroscopy + fractional curettage , hysterectomy and appendectomy ; other benign tumors included hydrosalpinx , ovarian corpus luteum cyst , mesosalpinx cyst , myoma , ovarian steroid cell tumor , ovarian thecoma , ovarian granulosa cell tumor , tubal serous glands fibroma ; value ; spls : single port laparoscopic surgery ; : no data . no remarkable difference was shown between the two groups regarding the median operation time ( 51 min vs. 52 min , p = 0.909 ) , median postoperative duration of hospital stay ( 3 days vs. 3 days , p = 0.168 ) and the median change in hemoglobin level ( 10 g / l vs. 10 g / l , p = 0.795 ) from preoperation to postoperation day 3 . notably , in spls groups , the median ebl and the anal exsufflation time were significantly less than those of the conventional group ( 5 ml vs. 10 ml , p < 0.001 ; 10 h vs. 22 h , p < 0.001 ) . there were no complications in either group , including peripheral visceral injury , incision hernia , or other postoperative morbidity . comparison of operative outcomes of the single port laparoscopic surgery group and the conventional group data are shown as median ( min , max ) . spls : single port laparoscopic surgery ; ebl : estimated blood loss ; pod : postoperative duration of hospital stay . based on visual inspection of the learning curve of the operation time from the 1 case to the 99 case in the spls group , the operative time gradually shortened as more cases were performed . a significant decrease in time was noted after 12 cases were done [ figure 4 ] . in the control group , operative time from the first case to 99 case in the single port laparoscopic surgery group . spls is a highly innovative technique that has been introduced in the field of minimally invasive surgery in recent years . although spls technique has been reported to perform tubal ligation for the 1 time in the 1970s , and ghezzi et al . succeeded in performing salpingectomy using spls in 2005 , the use of spls technique has not been widespread because of its technical challenges . fortunately , the innovation of operative techniques and design of new devices to overcome such difficulties have generalized the application of spls . several gynecologist groups have reported successful experience in diverse gynecological procedures using spls approaches such as laparoscopic - assisted vaginal hysterectomy and total laparoscopic hysterectomy . we performed a retrospective study to assess the use of spls versus conventional laparoscopy for the treatment of benign adnexal masses during the same period . considering that the patients enrolled in our study . this might lead to the difference of the baseline characteristics and the variety of diseases between the two groups . according to our data , the median age of the patients from both groups were equal ( 32 years vs. 32 years , p = 0.055 ) , but the variation in the spls group was slightly smaller than that of the conventional group ( [ 11 years , 58 years ] vs. [ 15 years , 73 years ] , p = 0.055 ) . moreover , the size of the ovarian tumor in the spls group was slightly smaller ( 49.0 mm vs. 55.5 mm , p = 0.064 ) . however , there was no statistically significant difference between the two groups regarding these two indicators . besides , from clinical experience , factors including body mass index , history of previous abdominal surgery and severity of pelvic adhesion were considered more relevant than age itself to the surgical outcomes and complications . furthermore , we believe the insignificant difference between the ovarian tumor size of the two groups was quite insignificant for experienced surgeons in clinical practice and would not have a dramatic influence on the main surgical outcomes . we concluded that no significant differences were observed regarding the operation time , perioperative decrease in hemoglobin level and postoperative hospital stay between the two groups . in the spls group , the median ebl was 5 ml and the median exsufflation time was 10 h. both outcomes were superior to those of the conventional group ( p < 0.001 ) . there were no cases of peripheral visceral injury or conversion to conventional laparoscopic surgery or laparotomy . additionally , there was a notable difference in the pathological types between the two groups ( p = 0.002 ) . in the spls group , mature cystic teratoma was the majority pathology type , comprising 27.8% of all cases . meanwhile , endometrioma made up 40.4% of the cases in the control group . this significant difference might have some influence on the surgical outcomes between the two groups , such as operation time and ebl due to pelvic adhesions that endometriomas commonly cause . however , there were no assumed differences in either the ratio of adhesiolysis performed during surgery or the operation time in both groups according to our study . the ebl in spls group was significantly less than that in the conventional group ( 5 ml vs. 10 ml , p < 0.001 ) , but it did not have any dramatic impact on the median changes in hemoglobin levels postoperatively ( spls : 10 g / l ; conventional : 10 g / l ; p = 0.795 ) . therefore , we consider the difference in the pathology types between the two groups to be minor as it did not influence the operative outcomes in our study . however , our short - term follow - up was not able to validate long - term complications between the two groups . several previous reports have reached similar conclusions that the surgical outcomes in benign adnexal surgeries did not differ significantly between spls and conventional laparoscopy . in our study the size of the ovarian mass was 70 mm 56 mm 51 mm . these positive results suggest that even in emergency cases like ovarian cyst torsion , spls might also have comparable surgical outcomes compared to conventional laparoscopic surgery . therefore , we would like to share our successful experience to draw more attention to single port procedures to manage benign adnexal masses . it is fundamental to maintain triangulation during spls procedures to avoid excessive instrument clashing and provide sufficient surgical field exposure . however , it is challenging because all of the laparoscopic working ports are placed through the same incision . in our experience , we used a 10-mm , 30-cm endoscope with a 30 angle to provide a wider range of vision and avoid collisions between surgical instruments compared with 0 endoscopes . we further used small trocars and prebent laparoscopic instruments to avoid collisions between instruments and to provide a wider range of motion . in spite of the above challenges , first , a single incision at the umbilicus might offer superior cosmetic outcomes by taking advantage of the concealing location of the umbilicus to hide the scar . second , it was easier to extract the specimen through a larger umbilical incision expanded by the spls wound retractor compared to the conventional 5-mm or 11-mm trocar incisions . third , by eliminating the trocars inserted in the bilateral lower quadrant the risks of complications including injury to the inferior epigastric vessels , subcutaneous hematoma as well as postoperative wound infection might be reduced . fourth , spls might be associated with less postoperative pain and the reduction of narcotic use due to few trocars being inserted . controversially , a larger umbilical incision may also cause a greater amount of postoperative pain . kim et al . demonstrated less postoperative pain score in the spls group for hysterectomy than that of the conventional group with comparable surgical outcomes . nevertheless , studies that compare levels of postoperative pain between the two groups are warranted . others include the costs of certain instruments , increased risk of complications including a hernia and possible increased postoperative pain associated with the larger size of the incision . however , the risk of a hernia does not appear to increase with the spls approach when the fascia is closed in a running , mass closure fashion . there were foreign researches reported that additional spls instruments would raise surgical costs . however , the spls ports we used were domestic products which hardly increased the surgical costs . still , the generalizability of spls technique requires additional effort and time to learn the operative skills . have demonstrated a remarkable decrease in operative time after 1015 spls were performed . according to our experience , proficiency in the learning curve as our surgeon is very experienced and has performed laparoscopic surgeries for more than 20 years , adopting spls technique for her was not as much of a challenge compared to junior physicians . thus , we did not expect a significantly prolonged operation time in the spls group . as a matter of fact , the operative time shortened as more cases were performed , compared to the control group , in which the operative time remained steady . in addition , the operative time in spls might also be shortened due to the easier extraction of the specimen as we mentioned earlier . as surgeons gain experience , the difference between the two groups regarding necessary operation time becomes insignificant . although we have concluded that spls approach has comparable surgical outcomes compared to conventional laparoscopic surgery regarding treating benign adnexal masses , as the operation type gets more complicated , spls approach might be confined by its complex technical challenge . therefore , despite that spls technique might have several advantages , it can not replace conventional laparoscopic approach in complex cases . studies that evaluate the feasibility and safety of spls for adnexal masses are not adequate . most of the previous studies have been limited to retrospective data and small samples of patients . in spite of the limitations of the study , including a retrospective data comparison with inevitable case selection bias and lack of postoperative pain score measurement and long - term follow - up , we have concluded that spls is a safe and feasible approach to treat benign adnexal masses . prospective studies with postoperative pain score measurement and long - term follow - up are needed to further assess the potential benefits and defects of spls .

background : single port laparoscopic surgery ( spls ) is an innovative approach that is rapidly gaining recognition worldwide . the aim of this study was to determine the feasibility and safety of spls compared to conventional laparoscopic surgery for the treatment of benign adnexal masses.methods:in total , 99 patients who underwent spls for benign adnexal masses between december 2013 and march 2015 were compared to a nonrandomized control group comprising 104 conventional laparoscopic adnexal surgeries that were performed during the same period . we retrospectively analyzed multiple clinical characteristics and operative outcomes of all the patients , including age , body mass index , size and pathological type of ovarian mass , operative time , estimated blood loss ( ebl ) , duration of postoperative hospital stay , etc.results:no significant difference was observed between the two groups regarding preoperative baseline characteristics . however , the pathological results between the two groups were found to be slightly different . the most common pathological type in the spls group was mature cystic teratoma , whereas endometrioma was more commonly seen in the control group . otherwise , the two groups had comparable surgical outcomes , including the median operation time ( 51 min vs. 52 min , p = 0.909 ) , the median decreased level of hemoglobin from preoperation to postoperation day 3 ( 10 g / l vs. 10 g / l , p = 0.795 ) , and the median duration of postoperative hospital stay ( 3 days vs. 3 days , p = 0.168 ) . in spls groups , the median ebl and the anal exsufflation time were significantly less than those of the conventional group ( 5 ml vs. 10 ml , p < 0.001 ; 10 h vs. 22 h , p < 0.001).conclusions : spls is a feasible and safe approach for the treatment of benign adnexal masses . further study is required to better determine whether spls has significant benefits compared to conventional techniques .

I M Operative techniques Statistical analysis R D Financial support and sponsorship Conflicts of interest

PMC4535086

the world health organization ( who ) estimates that almost 30% of the world population lacks access to essential medicines and that the figure will rise to more than 50% in some countries of africa and asia . the cost of the pharmaceuticals is the main factor that hampers access to medicines and the governments in poor countries seem to be doing very little to counter this problem . the public sector availability of essential medicines was less than 50% in most of the countries of africa and asia . this is appalling in the face of increases in healthcare expenditure in most of the developing nations , mostly financed through secured loans by international development banks and consortia . healthcare expenditures have been growing in india , both in real terms and also when considered as a proportion of the gross domestic product ( gdp ) . however , even with this recent increase in healthcare spending , india 's expenditure on health is nowhere near that of oecd ( organisation for economic cooperation and development ) nations . the total public spending on healthcare in india accounted for only around 1.2% of gdp in 2012 , with the per - capita spending on health around usd 160 . this is a miniscule amount when compared against the oecd per - capita healthcare spending of usd 3,484 in 2012 . this shows that the healthcare spending in the country is set to rise further in the coming years and the healthcare industry is all set for a boom time . the cost of medicines and pharmaceuticals as a percentage of total healthcare spending has also been rising worldwide . it is the fastest - growing item in the healthcare budgets worldwide and it varies between 20 - 60% in various healthcare budgets of countries . by 2020 , the prescription drug market in united states of america is set to grow to usd 700 billion ( b ) and china will be usd 260 b. though no credible predictions about the indian pharmaceutical industry are available , it is quite safe to assume that indian pharmaceutical industry will also grow manifold . the growth of the pharmaceutical market worldwide and its increased share in total healthcare spending will reignite the age - old debate on how to balance the cost of innovation in drug research and universal access to the fruits of that research . the role of generic medicines in reducing the healthcare expenditure has been recognised for a long time . multiple studies have proven that saving through substitution of originator brands by cheaper generic medicines , savings in the range of 10 - 90% can be achieved . most national governments have been encouraging the use of generic medicines worldwide and many healthcare systems have policies of substituting expensive branded original medications with generic medicines . in the united states , generic substitution ( gs ) is an accepted practice and at the end of 2012 , almost 80% of all the prescriptions were of generic medications . this has resulted in a substantial moderation of expenditure growth in widely used drugs and significant savings to the economy . in the united kingdom , gs is now a standard practice in hospitals operated by the national health service ( nhs ) and medical schools have included generic prescribing as a part of their medical training . in india , the procurement price of essential medicines is generally lower than the mean international reference pricing ( irp ) but availability of these drugs in the public sector has always been a problem . the exorbitant cost of some of the commonly used medications in private pharmacies makes it inaccessible to majority of the poor . also , the difference between procurement prices and retail prices in case of some of the generic medicines , were as high as 28 times , which shows a very high margin of profit - taking in view of limited price control mechanisms . it is in this light , that the government revised the national pharmaceutical pricing policy in 2012 . it gave methods to calculate ceiling prices for drugs which are under the national list of essential medicines ( nlem ) which was modified in 2011 . it gave a formula for deciding the ceiling prices for drugs under nlem , using a market - based pricing ( mbp ) method , taking into account the prices of all manufacturers having a market share of more than 1% nationally . the drug price control order of 2013 was a follow - up to the national pharmaceutical pricing policy and gave the price ceiling for 348 drugs and over 600 formulations . however , the action was considered inadequate by many activists lobbying for cheaper drugs and they termed it as a sell - out to international pharmaceutical companies . the multiplicity of brands and manufacturers makes it difficult to decipher the actual market dynamics and the structural issues in the indian pharmaceutical industry . the complexity of the market and the intensity of the competition between companies in india have made the country a hub for manufacture of generic medicines , earning a sobriquet pharmacy of the developing world . this , along with a favorable governmental stance has made india a powerhouse in this field , bringing it into direct confrontation with certain developed nations where most of the big multinational pharmaceutical companies are located there have been many instances when the indian patents office and the supreme court of india effectively used certain flexibilities of the trade related aspects of intellectual property rights ( trips ) agreement of the world trade organization and also the safeguards embedded in the indian patents act . the compulsory licensing of sorafenib , a drug used in treatment of advanced liver and renal cancer and the rejection of patent application for imatinib , a drug used in the treatment of leukaemia , were considered as landmark decisions by many state and non - state organizations involved in pharmaceutical sector . considering the indian scenario , we can divide the brands into innovator brands ( ib ) , most - selling generics ( msg ) , and least - priced generics ( lpg ) . the ibs will be at the highest price point , followed by msgs and lpgs . a new category of generic drugs known as unbranded generics ( ub ) are also coming into the market now . these drugs are usually manufactured by not - for - profit organizations or are subsidised by certain non - governmental organizations ( ngo ) . though the price points of these different categories of drugs are different , their efficacies are comparable . this fact has been proved by multiple studies all over the world and it belittles the reasoning which goes behind differential pricing of the same drug . even though it has been proved that there is not much difference in efficacy between the above categories of drugs , physicians tend to prescribe drugs manufactured by highly - reputed companies . their trust is often misplaced as most of these leading companies market drugs manufactured by less - known manufacturers . pushpagiri medical college , which is a teaching hospital in kerala state of india partnered with a social organization , bodhana social service society , involved in poverty alleviation and income generation programmes , to start an urban health center with an objective to improve patient accessibility to cost - effective medical care . the urban health center serves a population of 10,000 , spread over 5 municipal wards of tiruvalla municipality and was intended as a model for cost - effective primary care . a comprehensive population survey was carried out before the start of the project and the health center started functioning in september 2014 . as a part of the initiative , a community pharmacy was opened to stock unbranded generic drugs manufactured by two non - governmental organizations . low - cost standard therapeutics ( locost ) , baroda and comprehensive medical supplies india ( cmsi ) , chennai were the two ngos providing us with the drugs which were needed at the community pharmacy . the drugs were provided to us at a nominal cost , after we provided an undertaking that the pushpagiri medical college is a charitable institution with no intention of making profits . also , the physicians working at the health center made a collective decision to prescribe all the drugs generically and the pharmacist was advised to dispense the cheapest generic brand . the drug inventory available with these not - for - profit manufacturers were fairly comprehensive when reviewed using the world health organization - health action international ( who - hai ) tool for quantifying availability of essential medicines . the who - hai tool is a validated method for measuring availability of drugs in a health system and includes 30 core medicines : 14 essential medicines for global burden of disease and 16 medicines specific to the who region [ table 1 ] . drug inventory of locost , baroda and cmsi , chennai : review using who - hai tool for who south east asian region the who - hai tool showed a drug availability of 73.3% for locost , baroda and 43.3% for cmsi , chennai . this is much better when compared to drug inventory in public hospitals in other parts of india , assessed using the same methodology . there are a multitude of companies and ngos manufacturing ub medicines and the drug inventory of a health system can be made comprehensive through a mixed purchase model where procurement is done from multiple vendors . similarly , unbranded generic drugs offered significant savings to the health system in terms of costs involved for procurement . when reviewed against the msbs , ub medicines were costing only a fraction of the maximum retail price ( mrp ) of msps [ table 2 ] . comparison of drug prices of most - selling brands and their generic counterparts : drugs identified by who - hai tool for who south east asian region the community pharmacy has been in operation since september 2014 , and stocks over 120 formulations manufactured by unbranded generic manufacturers . in addition , it also supplies lsg to augment the drug inventory of the pharmacy . there is a family physician and a general practitioner who run the center , apart from regular specialist visits from pushpagiri medical college hospital . the urban health center has an outpatient load of 20 - 25 patients a day within 6 months of starting operations . the staff from the center is providing services to 3 old - age homes and a few surrounding schools and the drugs from the community pharmacy is being used for free supply during the medical camps conducted by the department of community medicine . the patients and the physicians have responded positively to this novel initiative and the general acceptability has been found to be high , though objective studies to assess the same are yet to be done . the financial sustainability of the model is still unproven , and the urban health center along with the community pharmacy is being sustained with large subsidies provided by pushpagiri medical college and bodhana social service society . the cost of setting - up such a facility was around inr 500,000 , which includes the furniture , basic medical equipment , basic lab accessories , and first round of procurement for the community pharmacy and is exclusive of the capital expenditure on the building . the average monthly expenditure in running the health center , has been around inr 150,000 a month , including salaries , cost of consumables and medicines and exclusive of building rent and depreciation . the income earned by the center is around inr 40,000 , and there is an excess of expenditure over income to the range of more than inr 100,000 a month , which is subsidised by pushpagiri medical college and bodhana social service society . both the organizations are charitable societies run by a prominent religious group and the subsidies are meant to further their commitment to social causes . the community pharmacy concept faced the following key challenges : absence of intermediaries for drug procurement results in inordinate delays in transit , mainly on account of the tardy services rendered by private logistics companiesadvance payment in full has to be remitted to the bank accounts of these ngos for supply of drugs , which goes against the standard practice of procurement followed in hospitals . this has been an issue with the internal audit departmentthe difference between procurement price and the mrp is minimal and this is causing worries of long - term financial sustainability of the community pharmacy modelpackaging of the drugs is unattractive in some cases , resulting in difficulty to convince patients about the efficacy of the drugwe have faced difficulty in convincing some of the specialist doctors on the quality of the drug , despite providing ample literature proving the efficacy of unbranded generic drugs . absence of intermediaries for drug procurement results in inordinate delays in transit , mainly on account of the tardy services rendered by private logistics companies advance payment in full has to be remitted to the bank accounts of these ngos for supply of drugs , which goes against the standard practice of procurement followed in hospitals . this has been an issue with the internal audit department the difference between procurement price and the mrp is minimal and this is causing worries of long - term financial sustainability of the community pharmacy model packaging of the drugs is unattractive in some cases , resulting in difficulty to convince patients about the efficacy of the drug we have faced difficulty in convincing some of the specialist doctors on the quality of the drug , despite providing ample literature proving the efficacy of unbranded generic drugs . most of these apprehensions are related to quality of the product and the fear of losing patients . along with these unfounded concerns , poor patient acceptability due to various issues like poor packaging , lack of brand promotion initiatives , etc . , are affecting the extend of penetration of ub drugs in the country , even though india is becoming a lifeline for all developing countries in the supply of generic medicines . the government and the policy makers in india and other similar developing countries should focus on building the confidence of physicians and the patients regarding unbranded generic medications . the demand side management should include a multifaceted approach in which issues of different stakeholders are addressed and affirmative actions taken in favour of unbranded generic medicine manufacturers . another important issue is concerning the inherent deficiencies and implementation status of the drug price control order of 2013 . the said order has been criticised extensively for being myopic in its approach , as the number of formulations included is less than 20% of the whole pharmaceutical market . also , it gave ample space for pharmaceutical companies to tweak their marketing strategies by focussing on formulations and dosages not covered by the drug price control order . it also leaves out the important area of fixed - dose combinations ( fdcs ) , a potential loop - hole for the pharmaceutical companies to exploit fully . it is indeed distressing to note that more than 90% of the diabetic drug market is out of the purview of this order . the policy makers in the country needs to get a realisation that the share of drugs in out - of - pocket expenditure ( opp ) is around 80% in india and a tighter regulatory framework is needed to protect the consumers against exploitation . in the future , we intend to do a study on the perception about generic drugs , among the treating physicians and the patients who form the clientele of the community pharmacy . this can help us to understand the issues which affect the actual stakeholders and find means to improve the acceptability and penetration of generic medicines . also , after the yearly financial audit , we plan to do a cost - benefit analysis to objectively analyse the efficacy of the model in monetary terms .

the cost of pharmaceuticals , as a percentage of total healthcare spending , has been rising worldwide . this has resulted in strained national budgets and a high proportion of people without access to essential medications . though india has become a global hub of generic drug manufacturing , the expected benefits of cheaper drugs are not translating into savings for ordinary people . this is in part due to the rise of branded generics , which are marketed at a price point close to the innovator brands . unbranded generic medicines are not finding their way into prescriptions due to issues of confidence and perception , though they are proven to be much cheaper and comparable in efficacy to branded medicines . the drug inventory of unbranded generic manufacturers fares reasonably when reviewed using the world health organization - health action international ( who - hai ) tool for analysing drug availability . also , unbranded generic medicines are much cheaper when compared to the most selling brands and they can bring down the treatment costs in primary care and family practice . we share our experience in running a community pharmacy for an urban health center in the pathanamthitta district of kerala state , which is run solely on generic medicines . the drug availability at the community pharmacy was 73.3% when analyzed using who - hai tool and the savings for the final consumers were up to 93.1% , when compared with most - selling brand of the same formulation .

Introduction Rise of Generics Indian Pharmaceutical Industry A Model Community Pharmacy: Experiences from South India The Way Forward

PMC3213722

bronchopleural fistula has a large number of etiologic factors ( infective , traumatic , or neoplastic ) and the even rarer esophageal - pleural fistula also has quite a large number of similar etiologies . esophageal ruptures usually present with mediastinitis or mediastinal abscess and can rarely present with esophago - pleural fistula without any mediastinal abscess . the diagnosis of esophago - pleural fistula without any mediastinitis is often delayed and difficult . bronchopleural fistula can be suspected on a plain radiograph and can be detected on ct scans . small and peripheral fistulae can close spontaneously and often do not need any surgical intervention apart from the intercostals tube drainage . we describe the imaging findings and procedure of endoscopic glue injection in a case of combined esophageal - pleural and bronchopleural fistula after penetrating thoracic trauma . a 40-year - old man came to the casualty with a thoracic bullet injury . at presentation , he had loss of power in both the lower limbs and dyspnea . the supine plain radiograph of the chest taken in the casualty revealed a bullet in the left hemithorax and diffuse - increased opacity of the left hemithorax . an axial ct scan of the dorsal spine revealed a fracture of the sixth dorsal vertebra with comminution of fragments and canal compromise ( not shown ) . the bullet was seen to lie in the left pleural cavity and a left - sided pleural effusion was present . the patient was taken for orthopedic procedure including posterior fixation and retrieval of the bullet from the pleural cavity . following the surgical procedures he was kept in intensive therapy unit where in the post - op days he was detected to have reduced air entry on the left side of the chest . once oral feeding was started , leakage of food material through the intercostals tube was noted and an esophago - pleural communication was suspected . a contrast - enhanced computed tomography of the thorax was performed after giving oral iodinated nonionic contrast . axial section of mediastinal window at the level of carina showed left hydropneumothorax and compression collapse of the left lung lower lobe . orally administered iodinated contrast was seen to accumulate in the left pleural cavity , suggesting an esophago - pleural fistula . the iodinated contrast was seen to follow a long irregular track in the mediastinum communicating the mid - thoracic esophagus to the pleural cavity and running in close proximity to the left main bronchus [ figure 1 ] . cervical esophagostomy , cervical esophageal exclusion , and feeding jejunostomy were performed as a therapeutic procedure . in the postoperative period he was detected to have a continued air leak and bronchoscopy bronchoscopy revealed a rent in the anterior wall of the left main bronchus ( 2 mm in diameter ) [ figure 2 ] . considering the general condition of the patient a bronchoscopic closure of the fistula was planned for . under general anesthesia , using a 4 fr angiographic catheter , 0.5 ml of n - butyl - cyanoacrylate glue ( nectacryl , dr . reddy 's laboratories , hyderabad , india ) mixed with 0.5 ml of iodized oil ( lipiodol ; guerbet , aulnay - sous - bois , france ) was injected in the fistulous tract [ figure 3 ] . immediately after closure of fistula , the pneumothorax reduced . follow - up imaging after 1 month revealed resolution of the hydropneumothorax and only small residual left lower lobe superior segment collapse . a bronchoscopy done at this point of time revealed granulation tissue lining the mucosal aspect of the fistula , which had sealed . the contrast swallow study was normal and he was started on oral feed ; esophagostomy and feeding jejunostomy were closed . six - month follow - up imaging revealed complete resolution of the hydropneumothorax [ figure 4 ] and good expansion of the left lung . axial cect scan mediastinal window after oral contrast showing a contrast lined mediastinal track connecting the thoracic esophagus and the pleural cavity . the track lies in close relation to the left main bronchus bronchoscopic image at the level of carina showing the fistulous opening in the left main bronchus bronchoscopic image showing the appearance of the tract after injection of the glue , the glue cast is seen to project into the bronchial lumen as a glistening white material axial cect scan mediastinal window 6 months after glue injection revealed dense glue cast in the mediastinal fistulous track . bronchopleural fistula can arise as a result of infective etiology ( rupture of a lung abscess , tuberculosis or fungal infection ) , traumatic ( iatrogenic in the form of lung biopsy or chest tube insertion or accidental ) , by invasion by a malignancy or after pneumonectomy . bronchopleural fistula has an incidence of 2% in cases of lung resection and in up to 10% cases after pneumonectomy . esophageal - pleural fistula can occur as a result of iatrogenic trauma ( following endoscopy ) , barotraumas , following empyema thoracis , in esophageal malignancies , tuberculosis . the esophagus is closely related to the left pleura at the thoracic inlet , to the right pleural space below the level of the azygos arch , at the level of gastroesophageal junction , the esophagus is closely related to both the pleural surfaces . the possibility of a direct fistula and mediastinitis depends on the location of esophageal injury and also the amount of mediastinal fat . , only one case has been reported in the english literature where the fistulae developed after pneumonectomy . the simultaneous presence of esophageal and bronchopleural fistula in our case was due to a long tract created by the path of the bullet in the mediastinum injuring the esophagus as well as the wall of the left main bronchus . a chest radiograph can be a useful screening modality for a bronchopleural fistula ; however , ct can demonstrate the site of fistula and can aid in surgical treatment by delineating the relation with the major airways . traumatic bronchopleural fistulae ( in penetrating trauma ) if small and distal , can be managed conservatively with intercostal tube drainage . those that do not resolve spontaneously can be treated via thoracoscopy using stapling devices and newer techniques such as intrapleural and intrabronchial administration of fibrin glue . blunt chest trauma usually results in main airway injury and does not lead to a pleural involvement . the surgical procedure for a large proximal fistula includes vascularized flap repair ( muscle , omentum , diaphragm , or pericardium ) . endoscopic closure of a bronchopleural fistula has been attempted using a variety of materials including fibrin , autologous blood clots , doxycycline , detachable balloons , silicon rubber plugs , glue , and metallic coils . in 1977 , hartmann and rausch reported closure of a postoperative peripheral fistula after two bronchoscopic applications of methylcyanoacrylate . described an endobronchial closure of a chronic bronchopleural cutaneous fistula with glue and metallic coils delivered through angiography catheters under fluoroscopic guidance ; performed under topical anesthesia . glue solidification of glue takes around 10 seconds when it comes in contact with the mucosa . initially the glue closes the fistula by mechanical sealing ; which is followed by inflammatory reaction and fibrosis which causes scarring and permanent fistula closure . in our case the glue injected in the tract formed a cast soon after the injection and sealed the fistula immediately ; evident by the immediate reduction of pneumothorax . in the course of time granulation tissue followed by fibrous tissue formed and the redundant part of glue cast lying within the bronchial lumen was shed off . fibrosis and scarring of the track helped seal the mucosal opening of the fistula . in conclusion , combined esophageal - pleural and bronchopleural fistula following penetrating trauma is a rare entity and the treatment included two - step approach ; first closure of the esophageal fistula by esophageal exclusion followed by endoscopic injection of glue for closure of bronchopleural fistula . endoscopic closure of fistula saved the patient from the morbidity associated from another thoracic surgery .

bronchopleural fistulas can occur from a number of causes ( infective , traumatic , or neoplastic ) . combined esophageal - pleural and bronchopleural fistula is not a common entity and previously has been reported after pneumonectomy . we describe the imaging findings and procedure of endoscopic glue injection in a case of combined esophageal - pleural and bronchopleural fistula after penetrating thoracic trauma . the treatment included esophageal exclusion for esophageal - pleural fistula , followed by endoscopic injection of glue for closure of bronchopleural fistula . the fistulae were completely sealed .

INTRODUCTION CASE REPORT DISCUSSION

PMC4506531

an internal hernia congenital or acquired is a protrusion of viscera through an opening in the peritoneum or mesentery . internal hernias are the etiology of < 2% of intestinal obstructions , with paraduodenal hernias being the most common type of congenital internal hernia . paraduodenal hernias result from the failure of mesenteric fusion with parietal peritoneum and malrotation of midgut and subsequent development of potential space in the left paraduodenal fossa . symptoms associated with paraduodenal hernias are generally nonspecific ; therefore , diagnosis is most commonly due to incidental findings on imaging or at laparotomy / laparoscopy . due to the fact that paraduodenal hernias can lead to bowel obstruction , ischemia and perforation with a high mortality , an elevated index of suspicion paired with diagnostic imaging / procedure is essential to making a timely and correct diagnosis [ 5 , 6 ] . in this review , we discuss the clinical presentation and management of small bowel obstruction secondary to a left paraduodenal hernia ( lpdh ) . a 69-year - old caucasian male was admitted to the hospital due to abdominal pain of 20 h duration . achy , constant , diffuse and with no radiation . associated nausea and similar episodes of pain over the past 2 years were noted . patient denied vomiting , fever , weight loss or change in bowel habits or urination . only significant past medical and surgical history being prostate cancer treated with radiation . on examination , the patient appeared in mild distress and vital signs within normal limits . physical exam revealed a moderately distended abdomen with an initially incarcerated umbilical hernia . the hernia was able to be manually reduced , which resulted in slight decrease of pain . hematological and biochemical studies were within normal limits , and a computed tomography ( ct ) scan demonstrated borderline dilated loops of proximal small bowel ; however , no transition point was identified . after discussion with a radiologist , a partial small bowel obstruction secondary to possible lpdh was confirmed ( fig . 1 ) . the patient was taken to the operating room within 12 h of hospital admission where a diagnostic laparoscopy was performed . while running the bowel , a hernia sac was discovered arising from a defect to the left of the fourth part of the duodenum , consistent with a lpdh ( fig . an aggregation of intestinal loops was herniated through the defect into the fossa of landzert . the bowel was then easily reduced using bowel graspers , and a scar was noted to be tacking the orifice to the mesentery , trapping the first part of the jejunum . remaining visibly dusky , a small midline laparotomy was performed to allow tactile examination of the bowel . after the mild ischemia was observed to have reverted and no further obstruction was ensured , the bowel was returned to the abdominal cavity and the lpdh orifice was closed primarily by fixing the root of the mesentery to the posterior parietal peritoneum . the patient was discharged on the seventh postoperative day , with instructions for resumption of normal daily activities . internal hernias are a rare pathology of intestinal obstruction , accounting for < 2% [ 2 , 4 ] . paraduodenal hernias , being the most common type of congenital internal hernias , are responsible for 1% of small bowel obstructions . lpdhs have been referred to by various terms : treitz retroperitoneal hernia , hernia of the fossa of landzert , mesentericoparietal hernia of logace and hernia into the descending mesocolon of callader . overall , a lpdh is three times more common than a right paraduodenal hernia ( waldayer 's hernia ) [ 2 , 5 , 7 ] . lpdh occurs when small bowel , usually jejunum , prolapses posteroinferiorly into the defect of the left paraduodenal fossa . landmarks of the fossa of landzert being left of the fourth part of the duodenum , anterior to the posterior peritoneum and posterior to the inferior mesenteric vein and left branches of the middle colic artery [ 3 , 8 , 9 ] . in congruency with presented case , median age range at diagnosis is fourth and sixth decades , and the male to female ratio is 3:1 . in this review , patients typically presented with signs and symptoms compatible with bowel obstruction secondary to incarceration , strangulation or necrosis . abdominal pain and associated symptoms being generally nonspecific , with 50% recounting recurring abdominal pain and occasionally a palpable mass [ 2 , 5 ] . most effective diagnostic tool for lpdhs is a ct scan , with typical appearance being an encapsulated sac containing dilated small bowl loops at the duodenojejunal junction with mass effect compression of the posterior stomach and distal duodenum . additionally , engorgement and distention of the mesenteric vessels will commonly shift the mesenteric trunk to the right and displace the transverse colon downward [ 1 , 4 ] . patients with a lpdh have a 50% lifetime risk of hernia incarceration with 2050% mortality for acute presentations ; therefore , after the diagnosis of a lpdh , operative management is recommended regardless of symptoms . in this review , laparoscopic and laparotomy repair is documented to be appropriate , using basic principles of hernia repair reduction of contents , restoration of anatomy and repair of the defect primarily or with mesh . alternative techniques of widening the hernia orifice and division of the inferior mesenteric vessels at the orifice can be implemented in cases where defect prevents repair using sutures or if reduction of contents from the fossa is a challenge [ 2 , 3 , 5 , 6 ] . intestinal obstruction secondary to an internal hernia is a rare entity ; however , delayed diagnosis and surgical intervention may result in significant morbidity and mortality [ 2 , 4 , 6 ] . therefore , in accordance with the above - noted literature and the surgical and clinical success of this case , it is important to include a paraduodenal hernia in the differential diagnosis of a patient presenting with small bowel obstruction and no history of abdominal surgery .

an internal hernia congenital or acquired is a protrusion of bowel through an opening in the peritoneum or mesentery . internal hernias are the etiology of < 2% of intestinal obstructions , with paraduodenal hernias being the most common type of congenital internal hernia . we report a case of a left paraduodenal hernia ( lpdh ) combined with partial small bowel obstruction in a 69-year - old male with recurrent abdominal pain of 2 years duration and no previous abdominal surgeries . an abdominal computed tomography scan showed an agglomeration of small bowel loops in the left upper quadrant but failed to yield a clear diagnosis . surgical intervention provided definitive diagnosis and treatment of the lpdh . we additionally review the literature regarding anatomy , pathogenesis , diagnosis and treatment of this uncommon hernia . intestinal obstruction secondary to an internal hernia is a rare entity ; however , delayed diagnosis and surgical intervention may result in significant morbidity and mortality .

INTRODUCTION CASE REPORT DISCUSSION CONFLICT OF INTEREST STATEMENT

PMC3719145

the protein complex models of rep1:ggtase - ii ( protein data bank [ pdb ] i d 1ltx ) ( fig . 1 ) and that of rab7:rep1 ( pdb i d 1vg9 ) ( fig . the software yet another scientific artificial reality application ( yasara ) dynamics and structure ( yasara biosciences gmbh , vienna , austria ) ( v 10.12.1 ) was used for generating the molecular model of ternary complex of rab7::rep1::ggtase - ii and also for its molecular dynamic simulation study . the online q - sitefinder programme ( university of leeds , leeds , uk ) was used for identification of putative dbss over the ternary complex and pymol ( schrdinger , usa ) ( an open source software ) was used for the molecular visualisation . for phylogenetic analysis , 60 human rab proteins were obtained from uniprot ( http://www.uniprot.org ) and the softwares used for the study were clustalw and phylip ( university of washington , seattle , usa ) . the structure files of protein complex models of rep1::ggtase - ii ( pdb i d 1ltx ) ( fig . 1 ) and that of rab7::rep1 ( pdb i d 1vg9 ) ( fig . 2 ) were downloaded from pdb and the two complexes were joined using yasara by selecting the strands of rep1 molecule in both the complexes , a single complex was generated . since the rep1 molecule was repeated , one of the rep1 molecules in the combining complexes was deleted by individually picking up the peptide chain . the ternary complex of rab7::rep1::ggtase - ii thus obtained was subjected to solvation option of yasara to add water molecules . to study the solvation effects on side chains of the protein complex a null model pka giving the lowest root - mean - square deviation ( rmsd ) without any shift in value was taken , where pka values were set as 3.22 , 4.09 , 6.20 , 10.8 and 10.76 for asp , glu , his , tyr and lys residues , respectively . structure of protein complexes of rep1 and ggtase - ii ( pdb i d 1ltx ) . the constructed and solvated system of ternary complex of rab7::ggtase - ii::rep was subjected to energy minimization without any constraints using steepest descent method followed by simulated annealing method . particle mesh ewald method was employed to calculate the electrostatic interactions with a cut - off of 10 . simulation snapshots were taken every 10,000 simulation steps , i.e. , with a timestep of 2.5 fs , i.e. , 10,000 * 12.5=25 ps . the constructed ternary complex of rab7::ggtase - ii::rep was initially subjected to energy minimisation by amber force field , using dynamics or structure module of yasara . then , after clicking onto simulation force field , yamber99 force field was selected in yasara dynamics . with the selection being made , simulation was run with command - options macro and movie play macro and double - clicking the standard macro em_run.mcr. the molecular dynamics simulation was then carried out in yasara dynamics or structure , where a project directory was created and the structure of ternary complex of rab7::ggtase - ii::rep was stored after energy minimisation . the molecular dynamics simulation was carried out after selecting the saved target molecule and following the command - options macro and movie play macro and double - click the standard macro md_run.mcr. the simulation performance was increased ( when needed ) by increasing the simulation steps per screen update using the data obtained after simulations were analysed for trajectory projection . this was done by using this macro created a self - explanatory table and was saved in project directory with tab - file extension . the table was then imported in the data visualisation program ( ms - excel ) that included energies and rmsds from the starting structure during simulation . the standard analysis macro also calculated the time average structure , whose atoms had the b - factors ( calculated from the root mean square fluctuations ) during simulation and the minimum energy structure was saved as energymin.sce in the project directory . to find the putative dbss of rab::rep::ggtase - ii complex , q - sitefinder programme was used in the present study . the q - sitefinder web site ( www.modelling.leeds.ac.uk/qsitefinder ) was visited and the pdb file of the ternary complex was uploaded and job was submitted . the q - sitefinder programme uses several separate procedures to perform ligand binding site prediction such as ligandseek , liggrid and probe - clustering programme . the probe clusters were ranked according to their total interaction energies , with the most favourable being identified as the first predicted binding site . the clustering programme also calculated site volume , and identified the protein atoms that were within a defined range of cluster sites . the programme generated the 10 best predicted sites ( interconnected dots ) and any ligands ( coloured by atoms ) were displayed as result . predicted binding site were listed in the display sites ( atoms forming the binding sites on the protein ) window according to the likelihood ( energy ) of the cluster of probes ( site 1 , site 2 ) . all together 60 different human rab proteins , relevant to the present study , were analysed for evolutionary relationship by multiple sequence alignment ( msa ) of their amino acid sequences . the amino - acid sequences of these proteins were obtained from uniprot ( www.uniprot.org ) . these sequences were then arranged in fasta format for msa by clustalw . the output file ( .phy ) was imported to the phylip for constructing the phylogenetic tree using neighbour - joining method . the structure files of protein complex models of rep1::ggtase - ii ( pdb i d 1ltx ) ( fig . 1 ) and that of rab7::rep1 ( pdb i d 1vg9 ) ( fig . 2 ) were downloaded from pdb and the two complexes were joined using yasara by selecting the strands of rep1 molecule in both the complexes , a single complex was generated . since the rep1 molecule was repeated , one of the rep1 molecules in the combining complexes was deleted by individually picking up the peptide chain . the ternary complex of rab7::rep1::ggtase - ii thus obtained was subjected to solvation option of yasara to add water molecules . to study the solvation effects on side chains of the protein complex , electrostatic interactions were screened . a null model pka giving the lowest root - mean - square deviation ( rmsd ) without any shift in value was taken , where pka values were set as 3.22 , 4.09 , 6.20 , 10.8 and 10.76 for asp , glu , his , tyr and lys residues , respectively . structure of protein complexes of rep1 and ggtase - ii ( pdb i d 1ltx ) . the constructed and solvated system of ternary complex of rab7::ggtase - ii::rep was subjected to energy minimization without any constraints using steepest descent method followed by simulated annealing method . particle mesh ewald method was employed to calculate the electrostatic interactions with a cut - off of 10 . simulation snapshots were taken every 10,000 simulation steps , i.e. , with a timestep of 2.5 fs , i.e. , 10,000 * 12.5=25 ps . the constructed ternary complex of rab7::ggtase - ii::rep was initially subjected to energy minimisation by amber force field , using dynamics or structure module of yasara . then , after clicking onto simulation force field , yamber99 force field was selected in yasara dynamics . with the selection being made , simulation was run with command - options macro and movie play macro and double - clicking the standard macro em_run.mcr. the molecular dynamics simulation was then carried out in yasara dynamics or structure , where a project directory was created and the structure of ternary complex of rab7::ggtase - ii::rep was stored after energy minimisation . the molecular dynamics simulation was carried out after selecting the saved target molecule and following the command - options macro and movie play macro and double - click the standard macro md_run.mcr. the simulation performance was increased ( when needed ) by increasing the simulation steps per screen update using the data obtained after simulations were analysed for trajectory projection . this was done by using macro and movie option and playing the standard analysis macro . this macro created a self - explanatory table and was saved in project directory with tab - file extension . the table was then imported in the data visualisation program ( ms - excel ) that included energies and rmsds from the starting structure during simulation . the standard analysis macro also calculated the time average structure , whose atoms had the b - factors ( calculated from the root mean square fluctuations ) during simulation and the minimum energy structure was saved as to find the putative dbss of rab::rep::ggtase - ii complex , q - sitefinder programme was used in the present study . the q - sitefinder web site ( www.modelling.leeds.ac.uk/qsitefinder ) was visited and the pdb file of the ternary complex was uploaded and job was submitted . the q - sitefinder programme uses several separate procedures to perform ligand binding site prediction such as ligandseek , liggrid and probe - clustering programme . the probe clusters were ranked according to their total interaction energies , with the most favourable being identified as the first predicted binding site . the clustering programme also calculated site volume , and identified the protein atoms that were within a defined range of cluster sites . the programme generated the 10 best predicted sites ( interconnected dots ) and any ligands ( coloured by atoms ) were displayed as result . predicted binding site were listed in the display sites ( atoms forming the binding sites on the protein ) window according to the likelihood ( energy ) of the cluster of probes ( site 1 , site 2 ) . all together 60 different human rab proteins , relevant to the present study , were analysed for evolutionary relationship by multiple sequence alignment ( msa ) of their amino acid sequences . the amino - acid sequences of these proteins were obtained from uniprot ( www.uniprot.org ) . these sequences were then arranged in fasta format for msa by clustalw . the output file ( .phy ) was imported to the phylip for constructing the phylogenetic tree using neighbour - joining method . the molecular model of the ternary complex of rab7::rep1::ggtase - ii ( fig . 3)was obtained using yasara edit option and was subjected to solvation . while studying the solvation effects on the side chains of protein complex , it was observed that the predicted pka range of lys , asp , glu , tyr and his were 10.46 - 11.84 , 2.82 - 5.09 , 3.74 - 5.08 , 10.8 and 6.34 - 7.42 , respectively . the results thus obtained were in good accordance of assumed null model and was thus subjected to further energy minimisation programme and force field calculations . structure of rab7::rep1::ggtase - ii ternary complex . after running the energy minimisation program , the ternary complex was subjected to simulation up to 500 ps ( 0.5 ns ) . the different snapshots of simulating steps were saved with respect to set time - intervals ( fig . thus , generated from these snapshots , reveals a very clear image of rab7 , ggtase - ii and rep1 interaction . the trajectory was obtained for overall energy simulation of the ternary complex of rab7:ggtase - ii : rep for 500 ps and it revealed that overall energy stabilised after a peak of -6363401.4 kj / mol at 25 ps and tended to remain in plateau phase further for rest of the period ( fig . this reflected that simulation was achieved with stable energy for rest of the period ( 50 - 700 ps ) for the said ternary complex . almost , the similar trajectory was obtained for the plots of different energy contributors against simulation run time . the contribution due to steric parameters like bond , angle , dihedral angle and planarity was found maximum at 25 ps with the values 828405 , 341330.665 , 357593.243 and 2679.375 kj / mol , respectively , which stabilised further to a stationary phase for rest of the period ( 50 - 700 ps ) , similar to the trajectory pattern of overall energy curve ( fig . the contribution due to columbian charge and van der waal interactions was also in accordance to the above results and was maximum at 25 ps with the values of - 9042365.5 and 1193953.512 kj / mol ( fig . 5f and g ) . the plots tended to stabilise further and attain the plateau phase for rest of the period ( 50 - 700 ps ) . these trajectory patterns supports and validates the simulation profile of the ternary complex of rab7::rep1::ggtase - ii . ( a ) total energy versus time , ( b ) bond energy versus time , ( c ) angular energy versus time , ( d ) dihedral angular energy versus time , ( e ) energy of planarity versus time , ( f ) energy due to columbian charge versus time and ( g ) van der waal interaction energy versus time . the simulation model investigation of the ternary complex of rab7::rep1::ggtase - ii suggests that the c - terminus of rab7 has to be in completely extended conformation during prenylation to reach the active site of rabggtase - ii . the results obtained in the present study are in good accordance with previous investigations and has provided a good picture of molecular dynamics of ternary complex of rab7::rep1::ggtase - ii and the molecular events during interaction of these three proteins . all together of about 60 - 70 putative dbss were generated by q - sitefinder programme . to predict the dbs precisely , these sites were comparatively analysed using an inhouse perl algorithm . the comparative analysis resulted in construction of comprehensive probe based on clustering method of energy criteria . the probe was found to conjugate fit with the rab7::rep1 : : ggtase - ii ternary complex ( fig . 6b ) and thus was a good primary indication of the correctness of the generated comprehensive probe . ( a ) the probe generated from q - sitefinder s results analysis and ( b ) ternary complex of rab7::rep1::ggtase - ii with the consensus probe . the comprehensive probe was used to integrate the putative dbss being generated by different q - sitefinder programme , as mentioned earlier . after an iterative process of selection and omission of different predicted dbss based on statistical parameters , a total of 10 major pockets were identified as putative dbss on rab7::rep1::ggtase - ii complex ( fig . 7 ) . these pockets reflected the energy of interactions of different probes being generated by the different used algorithms , viz . ; carbohydrate binding propensity , drug - like compound binding propensity , interacting pocket size threshold of radius 3 in three - dimension . these 10 major putative dbss thus identified were then mapped onto the rab7::rep1::ggtase - ii ternary complex ( fig . 7 ) which revealed that a total of two major pockets of dbs ( one in the -barrel and one in -helical portion ) were located on the rep protein of rab7::rep1::ggtase - ii ternary complex . of these two dbs , one was found to be located within the major cavity produced at the interaction point of rab7-rep complex with ggtase - ii . the other eight major putative dbss identified were found to be located on ggtase portion of the ternary complex . the -strand of ggtase was found to have two major pockets of putative dbss while the -strand possessed the other six pockets of putative dbss . all these putative dbs mapped completely over the rab7::rep1::ggtase - ii ternary complex , which was an indirect evidence of completeness of the computational calculation . these results have paved the path for further investigation into assigning rigidity to these putative dbss and develop pharmacophoric patterns for the same . these pharmacophore can be further used for virtual screening of ligand databases ( viz . , pubchem , zinc ) and are being presently investigated in our lab , apart from generating combinatorial libraries . the drug binding sites mapped on the ternary complex of rab7::rep1::ggtase - ii . after performing msa of the selected 60 different human rab proteins and constructing the phylogenetic tree by neighbour - joining algorithm of phylip of the same , it was observed that human rab7a , rab7b , rab9a and rab9b share the common ancestor and are the nearest neighbours during the course of evolution . so , the molecular dynamics studies of rab7::ggtase - ii::rep ternary complex will also provide a good platform for further investigation of related human rab proteins such as rab7a , rab7b , rab9a and rab9b . rab7 and rab9 are related to each other being localised to late endosomes and thus the phylogenetic analysis in the present study is coherent with the earlier results.[2931 ] to conclude , we can say that the present study has produced a good insight into the molecular events occurring during the ternary complex formation of rab7::rep1::ggtase - ii . the study also draws its significance because the protein studied is a membrane protein whose structural analysis is difficult through experimental way . the present in silico study was a primary and initial attempt in this direction and has paved pathway for further in silico studies like identifying the potential drug targets by searching the putative dbss , generating pharmacophoric pattern , searching or constructing suitable ligand , docking studies .

the structure - function correlation of membrane proteins have been a difficult task , particularly in context to transient protein complexes . the molecular simulation of ternary complex of rab7::rep1::ggtase - ii was carried out to understand the basic structural events occurring during the prenylation event of rab proteins , using the software yasara . the study suggested that the c - terminus of rab7 has to be in completely extended conformation during prenylation to reach the active site of rabggtase - ii . also , attempt was made to find putative drug binding sites on the ternary complex of rab7::rep1::ggtase - ii using q - sitefinder programme . the comprehensive consensus probe generated by the program revealed a total of 10 major pockets as putative drug binding sites on rab7::rep : : ggtase - ii ternary complex . these pockets were found on rep protein and ggtase protein subunits . the rab7 was found to be devoid of any putative drug binding sites in the ternary complex . the phylogenetic analysis of 60 rab proteins of human was carried out using phylip and study indicated the close phylogenetic relationship between rab7 and rab9 proteins of human and hence with further in silico study , the present observations can be extrapolated to rab9 proteins . the study paves a good platform for further experimental verifications of the findings and other in silico studies like identifying the potential drug targets by searching the putative drug binding sites , generating pharmacophoric pattern , searching or constructing suitable ligand and docking studies .

MATERIALS AND METHODS Construction of model of ternary complex of Rab7::REP1::GGTase-II: Simulation of ternary complex of Rab7::REP1::GGTase-II: Analysis of simulation trajectory of ternary complex of Rab7::GGTase-II::REP: Identification of putative DBS of Rab7::REP1::GGTase-II: Phylogenetic analysis of different Rab proteins found in humans: RESULTS AND DISCUSSION

PMC3227348

-synuclein is a 140 amino acid protein that has been implicated in several neurodegenerative diseases , often referred to as synucleopathies , such as parkinson s disease ( pd ) , dementia with lewy bodies ( dlb ) , and multiple system atrophy ( msa ) . pd , in particular , is neuropathologically characterized by -synuclein aggregates and the loss of dopaminergic neurons within the substantia nigra . while a number of theories have been advanced to explain how -synuclein self - association is related to neuronal dysfunction , the precise relationship between -synuclein aggregation and cell death remains unclear.(6 ) consequently , understanding the structural basis of -synuclein self - association is of particular importance . although monomeric -synuclein is intrinsically disordered in aqueous solution and is therefore considered an intrinsically disordered protein ( idp ) , it can not be simply described as a random coil . for example , the average radius of gyration of a random coil that is 140 amino acids long is larger than the measured average radius of gyration for -synuclein obtained via small - angle x - ray scattering ( saxs ) experiments.(10 ) this suggests that -synuclein is , on average , more compact than the classic random coil . the amino acid sequence of -synuclein contains 11-residue imperfect repeats that are distributed among the highly basic n - terminal region of the protein ( residues 160 ) , and the hydrophobic nac region ( non - a component of -synuclein , residues 6195 ) . these repeats were proposed to form amphipathic -helices capable of interacting with different types of lipid structures . it was found that when -synuclein is bound to micelles , two helices can form . the first helix encompasses residues 337 and is therefore contained within the n - terminal region and the second helix is formed between residues 45 and 92 , a region that begins in the n - terminal region and extends into the nac region . other studies suggest that -synuclein can also form a continuous helix that begins in the n terminal and continues through to the nac region and that the precise form of the helical segment depends on the precise experimental conditions . for example , in a recent study it was found , using pulsed dipolar esr spectroscopy , that depending on the relative protein - to - detergent concentrations , -synuclein can adopt either a single extended helix form or the broken helix form , similar to the one previously described.(21 ) moreover , recent data further suggest that under physiologic conditions , -synuclein exists in a tetrameric form that has considerable helical content.(22 ) by contrast , -synuclein aggregation is characterized by an increase in -sheet content . atomic force microscopy and raman spectroscopy demonstrated that soluble -synuclein oligomers have reduced -helical content relative to protofilaments , and that the sheet content is relatively increased in protofilaments and filaments.(23 ) fiber diffraction of -synuclein fibrils further demonstrated the presence of cross- structure which is characteristic of amyloid fibrils.(24 ) consequently , the available experimental evidence suggests that -synuclein can adopt helical structures or extended structures depending on the binding partner and experimental conditions . a number of studies have constructed -synuclein ensembles , using a combination of computational methods and experiments , to better understand the nature of the unfolded state . some of these studies combine data obtained from nmr , pre , and conformational sampling to construct an appropriate ensemble . while these studies have provided insights into the accessible states of -synuclein in solution , there are still many unanswered questions regarding the unfolded state of this protein , including the precise role of secondary structure in the unfolded ensemble and the presence of long - range contacts , in particular . in addition , the recent observation that -synuclein can also form ordered helical tetramers in the native cell environment has not been addressed in the previous studies . in this work , we use a recently developed bayesian weighting ( bw ) algorithm to construct an ensemble for wild - type ( wt ) -synuclein.(30 ) data from nmr chemical shifts,(31 ) rdcs,(32 ) and saxs(33 ) experiments are used to guide the construction of the ensemble . an analysis of the ensemble ( 1 ) helps to clarify the role of secondary structure propensity and the different binding characteristics of -synuclein , ( 2 ) identifies potential aggregation prone structures within the ensemble , ( 3 ) clarifies the relationship between long - range contacts and aggregation propensity , and ( 4 ) provides insights into how the disordered monomeric protein can form tetrameric helical structures . we began by generating a relatively large structural library of energetically favorable conformations and then used a bayesian weighting ( bw ) algorithm(30 ) to assign weights ( or relative stabilities ) for each conformer in the library . ensemble is defined as a set of structures , { si } and a corresponding set of weights w = { wi } where wi is the weight ( or probability ) of structure si and iwi = 1 . for a given structural library , there are many possible ways to weight the different structures within the structural library , and each possible weighting scheme represents a different ensemble . the bw method assigns a probability to every possible weighting scheme , and hence every possible ensemble , that can be constructed from the structural library . parenthetically we note that , since some of the wi can be 0 , finding a correct weighting scheme also enables us to exclude structures from the structural library if they consistently lead to ensembles that are inconsistent with the experimental data . the probability of a given ensemble is calculated using methods from bayesian statistics as described in our previous work(30 ) and as reviewed in the methods . overall the probability of an ensemble is related to the agreement between the data predicted by the ensemble and the experimental data . in the bayesian formalism , we compute a probability distribution ( which we refer to as the posterior density ) over all possible ensembles , and this distribution is used to make statements about the conformational properties of -synuclein . since the posterior density is a multidimensional function , we summarize its properties in two ways . first , we calculate the average weight of each structure in the structural library using the posterior density function . the ensemble consisting of the structures { si } and these average weights , w = { wib } is called the bayes ensemble ; that is , it is the bayesian analogue of a best fit ensemble . of course , the average of a distribution may not be very informative if the standard deviation , a measure of uncertainty , is large . to reflect this , we use the distribution over ensembles ( the posterior density ) to calculate confidence intervals for conformational characteristics of -synuclein as a way of quantifying statistical uncertainty . note that the confidence intervals do not refer to a specific ensemble , but rather to the distribution over all possible ensembles that could be constructed from the structural library . an advantage of the bw formalism is that it provides a built in estimate of the uncertainty in the bayes ensemble . since agreement with experiment alone does not ensure that an ensemble is correct , such quantitative measures of uncertainty are important.(30 ) a further advantage of the method is that , even when the uncertainty in the bayes ensemble is relatively large , we can calculate error bars to quantify the uncertainty in any observable quantity that is calculated from the ensemble . first , we constructed a structural library of 100 000 energetically stable structures by breaking the protein into overlapping 8-residue segments and exhaustively sampling the conformational space of each segment using replica exchange molecular dynamics ( remd).(34 ) segments were then joined to form a structure of the full 140 residue protein . to reduce the number of conformations to a more manageable size , the structural library was pruned using a coarse clustering method to generate a set of 299 structures that largely preserves the structural heterogeneity that was present in the original structural library ( figure 1 ) . application of the bw algorithm to obtain the bayes weight for each structure yielded a bayes ensemble that agrees with measured nmr chemical shifts(31 ) ( figure 2a ) and rdcs ( figure 2b)(32 ) as well as saxs derived radius of gyration(33 ) ( ensemble average value 41 1 vs experimentally determined value of 40 2 ) . these data demonstrate that the bw algorithm accomplishes its goal of generating ensembles that agree with the input experimental data . comparison of experimental results with the corresponding calculated ( a ) chemical shifts and ( b ) rdcs . calculated root mean square error ( rmse ) for the chemical shifts was found to be within accuracy provided by shiftx(66 ) . as discussed above , the bw method provides a built - in metric , called the uncertainty parameter , that quantifies our uncertainty in the bayes ensemble , and is analogous to the standard deviation of a gaussian distribution.(30 ) if it is likely that the bayes ensemble is correct , the uncertainty parameter approaches 0 . conversely , if it is unlikely that the bayes ensemble is correct , then the uncertainty parameter approaches 1 . in other words , as the uncertainty parameter approaches 1 , we can not say with any certainty that the constructed ensemble is correct . in the present case , , we can further quantify our uncertainty by computing confidence intervals for specific conformational characteristics . an analysis of the bayes ensemble provides additional information about the relative distribution of different conformer sizes that are accessible to the protein . as shown in figure 3 , the ensemble itself contains structures with radii of gyration that range from approximately 20 to 60 . to put these values into perspective , we note that the average radius of gyration for a globular folded protein containing 140 aa is approximately 15 , while the average radius of gyration for a random coil with the same amino acid length is approximately 52 .(35 ) the fraction of the ensemble with a radius of gyration near 20 is 0.09 ( 95% confidence interval 0.050.19 ) , while the fraction that has a radius of gyration greater than would be expected based on the random coil calculation is 0.17 ( 95% confidence interval 0.130.22 ) . this suggests that -synuclein samples structures that are nearly as compact as a globular protein of the same size in addition to structures that are more extended than that of the average random coil value . to assess the secondary structure content in the bayes ensemble , we used the stride secondary structure assignment algorithm,(36 ) to calculate the propensity of each residue , in every structure in the ensemble , to adopt one of three mutually exclusive classes of secondary structure : helix , strand ( also referred to as extended ) , and other ( see methods ) . analysis of individual structures within the ensemble reveals that the highest helical content is 20% while the highest strand content is approximately 28% . of note , the highest weighted structures in the bayes ensemble have helical content less than 15% and strand content less than 25% ( figure 4 ) . nevertheless , the ensemble average secondary structure content is considerably less ; that is , the overall strand content is less than 11% and the helical content less than 2% ( table 1 ) . however , the ensemble average , which corresponds to the experimentally observed value , is in excellent agreement with estimated secondary structure content obtained from cd spectroscopy(37 ) ( table 1 ) . although the experimental error bounds and the confidence intervals from the bw algorithm are relatively large for the helical and strand content , both bw and cd spectroscopy agree that the protein has minimal helical and strand content . the inset is an expanded view of the data . in addition to the overall secondary content of the protein , we also computed the expected ( or ensemble average ) relative helix and strand propensities for each residue in the protein with their corresponding 95% confidence intervals ( figure 5 ) . on average , this region contains a highly conserved hexamer motif within the fifth 11-mer imperfect repeat , which is proposed to form amphipathic -helices . additionally , within the nac segment , the strand propensity is peaked in the immediate vicinity of residue 78 . interestingly , this region , nac(818 ) , has been experimentally determined to be the minimal toxic aggregate forming segment in -synuclein in vitro.(38 ) ensemble average secondary structure propensity . for each residue , we present the probability to adopt a helical structure ( orange area ) vs a strand structure ( blue area ) . the thickness of the lines corresponds to the 95% confidence interval . to further demonstrate that -synculein samples structures with varying amounts of secondary structure , we explicitly show four conformations in figure 6 . the n - terminal region is marked in blue , the nac region in red and the c - terminal region in yellow . in figure 6a c , three structures are shown that contain varying degrees of helical content in the n - terminal and nac region . figure 6a shows a structure containing a helix between residues 42 and 64 ; this helical conformation is in agreement with the contiguous helix model . figure 6b shows a structure that contains a helix in residues 7482 in the middle of the nac region and figure 6c presents two helices , one in the range 5262 and the other in the range 1524 . figure 6d shows a structure with significant strand content in the nac region , in particular residues 6894 . representative ensemble conformations . a sample of structures from the ensemble of -synuclein . in all structures , blue denotes the n - terminal region ( residues 160 ) , red denotes the nac region ( residues 6195 ) , and yellow denotes c - terminal region ( residues 96140 ) . a number of studies have used paramagnetic relaxation enhancement ( pre ) experiments to detect long - range contacts in -synuclein . these experiments allow for the detection of interactions between a paramagnetic group and nuclear spins of residues at a distance up to 25 away.(41 ) some of these studies argue that long - range contacts , especially involving the n - terminal ( residues 160 ) and c - termini ( residues 96140 ) , can be found in the unfolded ensemble of -synuclein . to determine whether our data are consistent with these observations , we computed the distribution of such long - range contacts from the bayes ensemble . for these calculations , we define a long - range contact between the n- and c - terminal regions to occur when the center of mass of the n - terminal region ( residues 160 ) and the center of mass of the c - terminal region ( residues 96140 ) are within 25 . we computed these center of mass distances for each structure and used these data to compute the distribution of such distances along with the associated confidence intervals ( figure 7 ) . figure 7 demonstrates that structures in the bayes ensemble span a wide range of n- to c - terminal distances , ranging from less than 25 to more than 125 . in addition , a significant fraction ( 0.14 with a 95% confidence interval of 0.040.23 ) of the ensemble has structures that place the center of masses of the n- and c - terminal regions within 25 of one another . the x - axis represents the distance between the center of mass of the n - terminal region and the center of mass of the c - terminal region . two low probability structures had n- to c - terminal distances higher than 125 and we therefore excluded this information from the distribution . since results from pre experiments correspond to ensemble averages , we also computed a residual contact map ( which is a function of the ensemble average number of long - range contacts per residue ) to better compare our results to the previous pre data ( figure 8a ) . this pseudoenergy difference map represents the stability of a long - range contact between two residues in the bayes ensemble compared to what one would expect from a random coil ensemble ( see methods ) . these data suggest that there is a distinct preference for forming long - range contacts between the c - terminal ( residues 120140 ) and the n - terminal region ( residues 161 ) and also between the c - terminal and the nac region ( residues 6170 ) . in essence , residues 120140 in the c - terminal region make contact with residues 170 , which encompass both the n - terminal region and the beginning of the nac segment . a less favorable contact forms between the nac region ( residues 8095 ) and the n - terminal region ( residues 130 ) ; that is , our data are in qualitative agreement with prior experimental observations made from pre data . in this regard , it is important to note again that our -synuclein ensemble was generated without incorporating any data from prior pre experiments , and therefore , no explicit distance constraints were used to construct the model . stabilized long - range contacts . ( a ) pseudoenergy values are calculated for each contact as in(pijensemble / pijrc ) . a contact is defined between residues where the c atoms are less than 25 apart . a large negative pseudoenergy ( in blue ) represents contacts that are energetically favorable in the ensemble compared to the random coil ensemble ( in units of kt ) . ( b ) for each residue , we calculated the contact that is associated with the lowest pseudoenergy along with the 95% confidence interval . the x - axis is the residue number and the y - axis is the position of the residue that forms the lowest energy contact . we used the following color code to depict the different regions of the protein : blue , n - terminal region ( residues 160 ) ; red , nac region ( residues 6195 ) ; and yellow , c - terminal region ( residues 96140 ) . in figure 8b , we show the most stable contact for each residue , along with the corresponding 95% confidence interval . the relatively small error bars for residues 2070 and residues 120135 suggests that the model is relatively certain about these particular inter - residue contacts . however , the large error bars between residues 7090 argues that the model is unsure about the inter - residue contacts in this region . these data complement the residual contact map in figure 8a ; for example , the residual contact map suggests that there is a relatively small preference for forming long - range contacts between the nac ( residues 8095 ) and the n terminal region ( residues 130 ) ; however , the uncertainty analysis ( figure 8b ) suggests that the model is very uncertain about this particular observation . given that a relatively small segment of -synuclein , consisting of residues 6878 , which is found in the nac region ( i.e. , nac(818 ) ) , was experimentally determined to be the minimal toxic aggregate - prone segment in -synuclein in vitro,(38 ) we explored the conformational preferences of this segment . structures that place this segment in a solvent exposed and extended orientation may be more likely to form toxic aggregates containing cross- structure . figure 9 shows the normalized solvent accessible surface area ( sasa ) of the nac(818 ) region versus the number of residues in that segment that are in an extended conformation , as identified by stride.(36 ) calculations of the sasa only included the atoms h cc o as this ensures that large sasa values identify structures that can form the intermolecular hydrogen bonds that are needed for cross- structure formation . the bayes ensemble contains several structures that place the aggregation prone segment , nac(818 ) , in a relatively extended and solvent exposed orientation . we define a residue as solvent exposed when it has a normalized sasa > 40% , as this cutoff has been used in previous studies and useful results were obtained.(42 ) in total , the fraction of structures that have the nac(818 ) segment in a relatively extended and solvent exposed orientation is 0.08 with a 95% confidence interval 0.030.12 . the sasa vs number of residues in an extended orientation for the nac(818 ) region . two relatively high weighted structures with an exposed and a significant extended content ( more than 3 residues ) for the nac(818 ) segments are explicitly shown . it has been postulated that the formation of long - range contacts in -synuclein may provide a mechanism to shield regions of the nac segment.(26 ) burying regions of the nac segment could potentially hinder the formation of cross- structure and the formation of toxic aggregates . to investigate the relationship between solvent exposure of the nac(818 ) segment and long - range contacts , we computed the sasa of the structures that have the center of mass of the n - terminal and c - terminal regions within 25 . we find that the majority of structures that have the aforementioned long - range contacts also place the nac(818 ) segment in a solvent exposed orientation ; that is , 65% of structures ( 28%-100% , confidence interval ) with long - range contacts have the nac(818 ) segment with a sasa > 40% . in a recent study , -synuclein was isolated from human rbcs in a tetrameric form and the cd spectrum of this tetramer was quite distinct from that of recombinant -synuclein obtained from escherichia coli.(22 ) indeed , the spectrum of the tetramer had minima at 208 and 222 suggesting that , on average , the tetrameric structure had considerable helical structure . our data suggest that monomeric -synuclein samples structures that have at most 20% helical content ( figure 4 ) . the structure with the highest helical content is shown in figure 10a . in general , the associated helix has a hydrophobic patch on one side ( figure 10b ) , that is akin to hydrophobic faces that have been observed in other proteins that form helical bundles . these data are consistent with a model where helical segments within structures in the unfolded ensemble associate via hydrophobic interactions to form a tetrameric structure . if self - association of preformed helical structures was the dominant mechanism underlying the formation of tetrameric structures , then the expected helical content of the -synuclein tetramers would be at most 20% . interestingly , using the cd spectrum of the tetrameric structure ( kindly provided by tim bartels and dennis selkoe ) , we obtain a predicted helical content of 29% , with an error of approximately 10% , using the program k2d . ( a ) structure of the conformation within the ensemble that has the longest helical segment ( expanded view of structure shown in figure 6a ) . as before , blue denotes the n - terminal region ( residues 160 ) ; red denotes the nac region ( residues 6195 ) , and yellow denotes c - terminal region ( residues 96140 ) . ( b ) associated helical wheel : orange , nonpolar residues ; green , polar uncharged residues ; blue , basic ; pink , acidic . more recently , wang et al.(48 ) were able to obtain nmr data on a tetrameric form of -synuclein that was purified from e. coli . weak ( i , i + 3 ) nuclear overhauser enhancements ( noes ) and secondary chemical shifts indicated helical propensity in residues 4103 . intermolecular pres , obtained using mixtures of -synculein with and without the spin label , suggested that the tetramer forms by the association of amphipathic helices formed within the region consisting of residues 50103 . it is important to note , however , that the nmr data are not consistent with a fully folded helix . instead , they suggest transient helical formation with an overall helical content of approximately 20% ( tom pochapsky , personal communication).(48 ) these data are qualitatively consistent with our model presented in figure 10 , which includes an amphipathic helix consisting of residues 4764 , and with our finding of -helical propensity throughout the n - terminal region . although monomeric -synuclein is intrinsically disordered in solution , it can adopt conformations that have varying secondary structure content depending on its environment.(9 ) a number of spectroscopic studies suggested that wt--synuclein in the presence of membranes can form helical structures , and two types of helical configurations have been observed : a continuous extended helix and two antiparallel helices separated by a short linker . by contrast , -synuclein was shown to acquire significant -sheet content when it self - associates in vitro.(23 ) the most ordered form of these aggregates are fibrils which were found to contain cross--sheet structures.(24 ) most recently , an -synuclein tetramer has been isolated from both human red blood cells and e. coli and it has been argued that this structure is the dominant form under physiological conditions . while initial reports suggested this structure was a folded tetramer rather than an unfolded monomer , it is important to note that the data from recent nmr studies suggest that the actual amount of structural order is fairly low in the tetramer and that there is only fractional helix formation.(48 ) these observations highlight the need to obtain an accurate structural ensemble that describes the accessible states of the protein . while a number of studies have generated ensembles for the unfolded state of -synuclein , the majority of these ensembles has not provided a detailed analysis of residual secondary content within the ensemble and has not addressed recent data on what has been described as a physiologically dominant helical tetramer.(22 ) more importantly , existing studies have led to contradictory observations . although one prior study suggested a small preference for residues 637 ( in the n - terminus ) to form helices compared to residues 103140 ( in the c - terminus),(25 ) another study did not find significant helical structure within the ensemble.(27 ) therefore , to further explore the nature of the unfolded state of -synuclein and to understand the differential binding characteristics of the protein , we constructed and analyzed an ensemble that represents the unfolded state of monomeric -synuclein in solution . as we have previously noted , the construction of models that adequately represent the unfolded state of a protein is inherently difficult for a number of reasons . first , there is the conformational sampling problem ; that is , sampling all possible conformations of even a modestly sized protein is intractable . nevertheless , the form of the underlying energy surfaces helps because the space of energetically favorable conformations is likely far less than the space of all possible conformations . some studies have shown that straightforward boltzmann sampling for some idps yield calculated observables that are in reasonable agreement with experiment , thereby suggesting that extensive sampling , by itself , is a plausible approach for generating representative ensembles for idps . however , while it is reasonable to apply such a direct sampling approach to relatively small proteins , the prospect of extensively sampling the relevant conformational space of a protein that is 140 residues in length ( at 300k ) is daunting . in this regard , a number of approaches that do not rely on direct boltzmann sampling of large proteins have been developed and useful insights have been obtained using these methods . in the present study , we use a fragment based approach to sample energetically favorable conformations of the entire 140 residue protein . the motivation for this approach arose from a prior study that demonstrated that sampling the conformational states of fragments from folded proteins may reproduce the backbone structure of that peptide s structure in the context of the entire protein.(55 ) in our method , the protein was divided into eight residue long overlapping segments and remd was used to sample the conformational space of each peptide . eight residue segments were chosen because this length corresponds , roughly , to the average persistence length of a polypeptide.(52 ) structures for -synuclein were generated by combining these overlapping segments , and subsequent energy minimization of each reconstructed conformer ensures that each structure corresponds to local energy minima on the potential energy surface of the protein . while the approach is computationally efficient , we recognize that focusing on sampling small peptides may limit the formation of long - range interactions within the final ensemble . to mitigate this , once the peptide fragments have been combined to generate the -synuclein sequence , the entire protein is energy minimized thereby allowing the different peptides to see one another . using the resulting structural library with the bw algorithm , we arrive at a bayes ensemble that : ( 1 ) contains conformations that correspond to local energy minima on the potential energy surface and ( 2 ) agrees with the available experimental data . nevertheless , while our approach is computationally very efficient , we recognize that other sampling approaches for the initial structural library ( e.g. , using different lengths for the peptide segments ) may lead to different structural libraries and this fact introduces some uncertainty in our analysis . one additional source of uncertainty is the inherent degeneracy of the problem of constructing a good ensemble , even after the precise structural library has been specified . given that the number of degrees of freedom ( i.e. , the number of energetically favorable conformations ) is typically much greater than the number of independent experimental observables , the problem of choosing , or weighting , a set of structures is inherently degenerate ; that is , there are many possible ways of weighting the structures that will agree with any given set of experimental observations.(30 ) to deal with these sources of uncertainty , the bw method calculates a probability distribution over the space of ensembles . this posterior density function naturally leads to a new metric , the uncertainty parameter , which quantifies our uncertainty in the bayes ensemble . this uncertainty parameter is akin to the standard deviation in a gaussian distribution and reflects the overall spread of the calculated probability distribution . the uncertainty parameter varies between 0 and 1 where a value of 0 suggests that the bayes ensemble is correct . by contrast , when the uncertainty parameter is nonzero , one can not be certain that the bayes ensemble is correct . however , in this latter instance , one can express values with the appropriate confidence intervals.(30 ) therefore , the method provides a rigorous means to quantify the overall uncertainty in the final results . the bw algorithm yields a bayes ensemble that is in agreement with data obtained using nmr chemical shifts,(31 ) rdcs,(32 ) and the radius of gyration as determined by saxs experiments.(33 ) surprisingly , we find that some conformers in the ensemble are nearly as compact as a folded globular protein with the same amino acid length . in addition , the bayes ensemble contains structures that have a radius of gyration that is larger than the average radius of gyration that would be expected from a 140 residue random coil . this highlights the fact that a single experimental value for the radius of gyration provides little insight into the full range of conformations that the protein can adopt in solution . two recent experimental studies suggested the existence of distinct classes of conformers describing -synuclein equilibrium . both studies argue that -synuclein contains a range of conformations , where some are quite compact and others are quite extended and random - coil like . our data are in agreement with these observations and quantify the range of radii of gyration within the ensemble . moreover , these studies highlight the fact that while the overall secondary structure content of the ensemble is negligible ( about 7% of the population was suggested to contain -like conformation ) , there are subpopulations of structures that have more significant helical and strand content , a finding in agreement with our observations . the resulting structural ensemble provides additional insight into the secondary structure propensities within different regions of the protein . we find that the bayes ensemble contains several structures that have helical segments of varying length in the n - terminal region , extending into the nac segment . in total , the helical regions span residues 192 ; that is , the segment that has been shown to adopt either a continuous helix or a broken helix in the presence of lipid membranes . these data are consistent with a model of lipid binding where interaction with the membrane stabilizes these helical segments leading to the formation of either a continuous or a broken helix , depending on the precise experimental conditions . in this sense , the presence of relatively short helical segments may serve as intermediates that enable fast and efficient binding to lipid membranes . these data are of particular importance because interactions between -synuclein , in its helical form , and membranes may play a role in cellular dysfunction in patients with parkinson s disease.(58 ) by contrast , on average , significant probability for extended structure is found throughout the -synuclein sequence . these data are in qualitative agreement with previous raman spectroscopic studies that suggest that the protein adopts an ensemble of rapidly interconverting secondary structural elements.(59 ) of particular interest is the region spanning residues 6878 in the nac segment because this has been shown to be the minimal toxic peptide that can also initiate -synuclein aggregation in vitro.(38 ) the probability of extended structure is relatively peaked around this region , thereby suggesting that this segment has an intrinsic predisposition to form extended structure that may initiate the formation of -sheet rich aggregates . however , in order to form intermolecular hydrogen bonds with other -synuclein molecules , this segment must be exposed to solvent . therefore , structures that place this segment in a solvent exposed and extended conformation may be more prone to form toxic aggregates . our analysis suggests that approximately 8% , with a 95% confidence interval of 312% , of the structures in the ensemble have the nac(818 ) segment in an extended and solvent exposed orientation . this suggests that the unfolded ensemble of -synuclein contains preformed conformations that can readily form -sheet rich toxic aggregates . in addition to these insights , our data further clarify the role of long - range contacts in the protein . previous studies that have constructed ensembles based on the results of pre experiments have found conflicting findings , even though many of these experiments were performed under similar experimental conditions . one study suggested that long - range interactions occur between residues 8595 of the nac and the c - terminal region ( specifically residues 110130).(28 ) other studies suggested the formation of long - range contacts between the highly charged c - terminus ( residues 120140 ) and the large hydrophobic center ( residues 30100 ) resulting in a hydrodynamic radius significantly smaller than that expected for a random coil structure . in another study , done under similar conditions , it was suggested that long - range contacts form between the n - terminus and the nac region in contrast to the previously mentioned studies.(27 ) therefore , while these data have provided new insights into the nature of the unfolded state of -synuclein in solution , they leave the precise role of any long - range interactions in the protein unclear . our data suggest that , on average , there are long - range contacts between the n- and c - termini of the molecule . interestingly , the bayesian estimates allow us to say with confidence that the n - terminal region and the first nine residues from the n - terminal portion of the nac make , on average , contacts with the c - terminal region of the protein , a result that is in qualitative agreement with prior studies . it has been suggested that these long - range interactions provide a mechanism that effectively shields the aggregation prone region , and thereby minimizes the extent of aggregation . however , a more detailed look at the actual distribution of structures within the ensemble ( as opposed to an analysis of the ensemble average data ) finds that most of the structures in our ensemble that contain long - range contacts between the n- and c - termini also place the nac(818 ) segment in a solvent exposed conformation . consequently , it is not clear that separation of the n- and c - termini is required to expose the most aggregation prone regions of the sequence . this claim is supported by recent pre experiments comparing wt -synuclein and a30p , e46k , and a53 t naturally occurring mutants , which were all shown to have a higher aggregation rate in vitro . results of this study suggest that a30p and a53 t mutants did not have a significant decrease in the n- and c - termini contacts . moreover , e46k presented an increase in these long - range contacts.(39 ) these data bring into question whether long - range contacts play a key role in regulating aggregation of -synuclein . we note that our model did not use any pre derived distance restraints . while pre - derived data have provided valuable information into the presence or absence of long - range contacts in several idps , it requires introducing a paramagnetic probe into the protein.(61 ) however , it may be that such probes alter the accessible states of the unmodified protein . in light of these observations , and the fact that some of the pre - derived results are contradictory , we did not explicitly use pre - derived data when building our ensemble . nevertheless , we obtain results that corroborate and clarify many aspects of the prior pre studies . in addition , we recognize that there may be additional contacts between the n - terminal , nac , and c - terminal regions , but we can not make statements about these interactions with confidence given the very wide error bars associated with residues in the more central region of the nac segment ( figure 8b ) . interestingly , our uncertainty in the precise contacts that involve the entire nac region is also reflected in the literature as the nac region is suggested to interact with the c - terminus in some studies while other studies suggest that it interacts with the n - terminus instead . in short , our data suggest that monomeric -synuclein samples structures that have at most 20% helical content and that some of these helices are amphipathic in character . these data are consistent with a model where tetrameric structures are formed via the interaction of hydrophobic patches on these amphipathic helices . indeed there are many examples in the literature of such four - helical bundle structures composed of amphipathic helices . independently proposed a model for the tetrameric state of -synuclein on the basis of nmr data in which transiently formed amphipathic helices interact in just such a manner.(48 ) our results argue that the unfolded state of -synuclein contains a heterogeneous set of conformations of both highly compact and extended structures , and that while the overall secondary structure content of these structures is low , there are regions that have a relatively high propensity for helical and extended structure . regions with a significant propensity for either helical or strand content may facilitate the formation of lipid - associated helical structures , helical tetrameric structures , and aggregates that are rich in -sheets . our results also provide quantitative estimates for the percentage of structures that are compact , have long - range contacts between the n- and c - termini , and that have the minimal toxic aggregation fragment of -synuclein that is in a position that is poised to make intermolecular beta strands . in sum , these data provide a comprehensive view of the unfolded ensemble of monomeric -synuclein in solution and explains how different ordered structures conformers can arise from this disordered protein . the sequence of -synuclein was divided into eight residue long segments resulting in 28 segments in total ( the c - terminal segment was five residues long ) . a similar protocol was used to describe k18 , an intrinsically disordered protein of comparable size , 130 amino acids long.(30 ) the size of the segments was chosen based on the average persistence length of a polypeptide.(52 ) conformations for segments of this length were shown to be successfully sampled using a replica exchange molecular dynamics ( remd(34 ) ) procedure.(63 ) each segment underwent remd with the eef1(64 ) implicit solvent model using charmm.(65 ) a total of 16 replicas , each at a different temperature , were used . segments were run for 10 ns , and structures were collected from the last 5 ns of the 298 k heat bath , allowing 5 ns of equilibration period . full length -synuclein conformations were generated by piecing together the segments one at a time , starting with the n - terminal segment . the segment to be added to the growing polypeptide chains was chosen from the cluster that had the most structural similarity in the overlapping region . the first residue coordinates of the overlapping segments were taken from the c - terminal region of the one segment and the two others from the n - terminal region of the adjoining segment . at the end of the procedure , the full length structure was subjected to 1000 steps of steepest descent minimization followed by 10 000 steps of adopted basis newton only structures with a negative energy were chosen for the structural library . following the process , we found the structural library generated was composed of structures that were mainly compact when comparing their radius of gyration ( rg ) to the one obtained by saxs experiments . therefore , the combined pre - energy minimization structures were used in additional energy minimization using an rg restraint . this process ensures that a wide range of conformations were generated . at the end of the process the structural library was reduced in size to 299 structures using our previously described pruning algorithm.(30 ) this number of structures was shown to be able to provide a good model for the k18 tau segment of comparable size ( 130 residues).(30 ) to obtain the sets of weights for the pruned structural library , we employed the bw algorithm as previously described.(30 ) in this method , one generates a posterior distribution which represents the probability of all possible weighting schemes over the 299 structures , given the available experimental data . experimental measurements used were c , c , c , and n chemical shifts,(31 ) n h rdcs,(32 ) and radius of gyration.(33 ) the carbonyl chemical shift value for residue 140 from the set of experimental data points was not used , as it was an extreme outlier from the other data.(31 ) to implement the bw method , we first need to calculate the corresponding chemical shifts for each atom , along with rdcs and the radius of gyration , in each structure . chemical shifts were calculated with shiftx,(66 ) and the radii of gyration were calculated with charmm.(65 ) the rdcs of each individual conformer in the ensemble was calculated with pales(67 ) based on a global alignment model , that is , using the entire protein structure . this is in contrast to a local alignment model , in which the rdcs are calculated from short segments of the protein . it has been suggested that one can reproduce experimental rdcs with a smaller number of conformers when using a local alignment model as compared to a global alignment model ; nevertheless , we were able to obtain good agreement with the experimental rdcs using the global alignment method with a relatively small number of highly populated conformers . the bw algorithm incorporates information from both the experimental errors and the errors associated with predictions for the experimental values of interest.(30 ) experimental errors were taken to be 0.3 ppm ( chemical shifts ) , 1 hz ( rdcs ) , and 2 ( radius of gyration ) , respectively . as prediction errors for chemical shift values have been rather extensively studied , they were also included in the expression for the posterior distribution . here , we provide a very brief review of the theoretical aspects of the bw framework ; for a comprehensive description see fisher et al.(30 ) formally , the posterior probability distribution conditioned on the observed experimental data is obtained from bayes rule : where fw ( w ) is a the prior probability distribution ( for brevity , the specific form will not be reproduced here ) , and fm|w(m|w ) is the likelihood function for the vector of experimental observations , m. we assume that the likelihood function can be decomposed as fm|w(m|w ) = fm|wrg ( m|w ) fm|wrdc(m|w ) fm|wcs(m|w ) where each of the components is ( multivariate)-gaussian . specifically , the likelihood functions arehere , the letter denotes an experimental error , denotes a prediction error , and is a factor for uniformly scaling the rdcs to account for uncertainty in the magnitude of alignment . the bayes estimate for the weight for each structure corresponds to the expected ( or average ) value of that structure s weight over the posterior distribution ; that is , wjb wjbw = dwwjfw|m(w|m ) . the uncertainty parameter is the average distance from the bayes weights , or w [ dw(w,w ) fw|m(w|m ) ] , where (w,w ) is metric on the space of weight vectors called the jensen - shannon divergence.(30 ) to calculate these expected values , samples are taken from the posterior distribution using a monte carlo algorithm with gibbs sampling.(30 ) each sample corresponds to a different weighting scheme over the 299 structures . a total of 100 million samples were generated as an equilibration period for the markov chain generated from the monte carlo algorithm . this was followed by an additional 1 billion samples , which constitutes the production run . we followed the running average of the posterior divergence to ensure that convergence was reached . to calculate the bayesian averages and the associated confidence intervals , we used 50 000 equally spaced samples from the 1 billion samples ; this reduces the overall computation time . for a given quantity ( e.g. , the expected solvent exposure of a given residue ) , we computed this quantity using the chosen samples , yielding 50 000 estimates for the value of interest . the 95% confidence interval was obtained by finding the lower bound that excluded the bottom 2.5% of the estimates and the upper bound that excluded the top 2.5% of the estimates . the residual contact map shown in figure 8a represents the stability of a long - range contact between two residues in our ensemble compared to what one would expect from a random coil ensemble . therefore , to compute the contact map , we first need to generate a random coil ensemble for -synuclein . we used the publicly available random coil ensemble posted in a web repository.(52 ) the ensemble contains 5000 structures ; we therefore randomly selected 299 structures to ensure the two ensembles are of the same size . the selection process was repeated 20 times in order to reflect the full ensemble and each measurement of interest was averaged over this collection . the random coil model used to form this ensemble uses statistical potential and excluded volume constraints;(52 ) no -synuclein experimental data was included in generating the ensemble . we clustered stride results of -helix , -helix , and 3 - 10 helix into a super class that we refer to as helix . in addition , we cluster isolated bridge and extended results into a second super class we named extended ( or strand ) . to have consistent definitions when comparing to the results obtained from cd spectroscopy ( which assigned a helix , strand , turn , and unstructured),(37 ) we grouped the turn and the unstructured assignments into one category called other . the solvent exposure surface area for each conformation was calculated using charmm.(65 ) the sasa of the entire protein was computed , but only data from the solvent exposure of the backbone atoms n h c co were used , since these represent atoms that are essential for the formation of cross--sheet interactions . the calculated sasa was normalized by dividing by solvent accessible surface of the backbone atoms when -synuclein is in a fully extended conformation . a residue is said to be solvent exposed when its normalized sasa > 40% , as this cutoff has been used in previous studies and useful results were obtained.(42 ) two plots were generated presenting probabilities calculated from the posterior distribution . the radius of gyration for each structure in the ensemble is calculated in charmm(65 ) using the n summation of the structures probabilities ( their weights ) in each bin comprises the probability of that bin . the 95% confidence intervals were then obtained using the 50 000 samples from the posterior distribution as outlined above . the histogram of n - terminal center - of - mass to c - terminal center - of - mass distances was generated in a similar fashion . distances were calculated in charmm(65 ) using the center of mass of n c c backbone atoms for residues 160 , the n - terminus , and the center of mass of n c c backbone atoms for residues 96140 , the c - terminus . structures were binned in bins of 25 , corresponding to the maximal distance defined for formation of long - range contacts and again the 95% confidence intervals were then obtained from the 50 000 samples from the posterior distribution . to generate the helical wheel amino acid sequences taken from the conformation with the longest continuous helical structure were input to the helical wheel program to generate the associated diagram .

given that -synuclein has been implicated in the pathogenesis of several neurodegenerative disorders , deciphering the structure of this protein is of particular importance . while monomeric -synuclein is disordered in solution , it can form aggregates rich in cross- structure , relatively long helical segments when bound to micelles or lipid vesicles , and a relatively ordered helical tetramer within the native cell environment . to understand the physical basis underlying this structural plasticity , we generated an ensemble for monomeric -synuclein using a bayesian formalism that combines data from nmr chemical shifts , rdcs , and saxs with molecular simulations . an analysis of the resulting ensemble suggests that a non - negligible fraction of the ensemble ( 0.08 , 95% confidence interval 0.030.12 ) places the minimal toxic aggregation - prone segment in -synuclein , nac(818 ) , in a solvent exposed and extended conformation that can form cross- structure . our data also suggest that a sizable fraction of structures in the ensemble ( 0.14 , 95% confidence interval 0.040.23 ) contains long - range contacts between the n- and c - termini . moreover , a significant fraction of structures that contain these long - range contacts also place the nac(818 ) segment in a solvent exposed orientation , a finding in contrast to the theory that such long - range contacts help to prevent aggregation . lastly , our data suggest that -synuclein samples structures with amphipathic helices that can self - associate via hydrophobic contacts to form tetrameric structures . overall , these observations represent a comprehensive view of the unfolded ensemble of monomeric -synuclein and explain how different conformations can arise from the monomeric protein .

Introduction Results and Discussion Conclusion Methods

PMC3075996

home bleaching is a popular and convenient treatment to improve the appearance of vital teeth.1 during this procedure , whitening gel is deposited on moulded acrylic trays that allow the solution to act indistinctly over both enamel and pre - existing aesthetic restorations . many investigations have shown that low - concentration bleaching substances may damage these structures13 and thus increase their susceptibility to staining and bacterial adhesion.46 major colour improvements are sometimes enthusiastically desired by patients , whereas the adverse effects of whitening procedures may be unintentionally downplayed by professionals . recently , manufacturers released a flood of high - concentration bleaching gels in the market . these gels are claimed to increase the effectiveness as well as longevity of at - home treatments . as expected , initial in vitro studies reported significant alterations to enamel7,8 or composite resins.2,9 however , these results were not confirmed by the scant evidence originating from clinical investigations.10,11 since the effects of whitening substances have been evaluated separately and in a great diversity of regimens , it is difficult to extend those incompatible evidences to aesthetically restored teeth . thus , this prospective clinical study aimed to assess simultaneously the effects of 16% carbamide peroxide on the external topography of enamel and composite resin . this study was initiated only after obtaining suitable approval from the local ethics committee , which is in conformity with the declaration of helsinki ( doh ) . all molars included fit the following criteria : 1 ) arising from different donors and 2 ) without structural damage , signs of hypomineralisation ( white spots ) or prior contact with saliva . after cleaning , teeth were individually positioned in a sectioning machine equipped with a water - cooled 300-m thick diamond saw ( minitorm , struers a / s , copenhagen , denmark ) . after that , the remaining crows were equidistantly sectioned in the mesiodistal and buccolingual directions to provide four enamel fragments ( 4 mm x 4 mm and 2 mm thickness ) each . the pieces obtained were moulded into a light regular - set silicon ( virtual light - body , ivoclair vivadent , so paulo , sp , brazil ) , and the moulds were filled with increments of a micro - hybrid composite resin ( ea2 opallis , fgm dentscare ltda , joinville , sc , brazil ) . moulds were sequentially polymerised over 20 seconds using a light - emitting diode unit set between 460 and 480 mw / cm ( ultra - blue i5 , dmc vasconcellos , so carlos , sp , brazil ) in order to produce enamel - like composite resin specimens . the resulting 40 specimens ( 20 of composite resin ) were then individually placed at the end of polyvinyl chloride cylinders ( 3/4 inch diameter and 20 mm high ) , which were filled with chemically - activated acrylic resin ( technovit 4000 , heraeus kulzer , wehrheim , germany ) . after 24 hours , the exposed side of each embedded fragment was flattened and polished by sequential use of wet 400- , 600- and 1200-grit silicon carbide papers ( norton,guarulhos , sp , brazil ) adapted in a slow - speed polishing machine set at 150 rpm ( ballerup , struers a / s , copenhagen , denmark ) . the finished specimens were then removed from blocks and ultrasonically cleaned for 5 minutes before being sterilised and stored in distilled water ( 370.5c ) until use . all volunteers presented the following inclusion criteria : absence of caries and gingivitis , no history of xerostomia , no previous bleaching therapy and willingness to understand and sign a consent form . participants had their maxillary right teeth cleaned with a rotary toothbrush and dentifrice before isolation with a rubber dam and stainless steel clamps . the buccal sides of the right upper second premolar and first molar were etched with 37% phosphoric acid ( dentsply , petrpolis , rj , brazil ) for 30 seconds and immediately washed with running water for 60 seconds . to fix the enamel and composite resin randomly on these surfaces , a two - step adhesive ( adper scotchbond , 3 m , sumar , sp , brazil ) was brushed and light - polymerised for 15 seconds before an increment of low - viscosity composite resin ( natural flow , dfl , jacarepagua , rj , brazil ) was used to glue the specimens on teeth using light - polymerisation for 40 seconds . these same procedures were performed on the adjacent lower right teeth to fix the enamel and composite resin on specimens not undergoing the bleaching process . an upper jaw impression was taken of each participant with alginate materials ( jeltrate , dentsply , petrpolis , rj , brazil ) , and impressions were poured with type ii dental stone ( pasom , so paulo , sp , brazil ) to fabricate a master cast . a 0.5 mm - thick wax layer was applied on the buccal face of each cast 1.5 mm away from the gingival margin to fabricate reservoirs for the bleaching gel . soft trays ( cristal , bio - art , so carlos , sp , brazil ) were made using a heat / vacuum tray - forming machine ( plasvac p7 , bio - art , so carlos , sp , brazil ) . the trays were tested and trimmed to fit the gingival margin of each volunteer . the participants were instructed to use the tray at the upper arch and to keep the bleaching product ( whiteness perfect 16% , fgm dentscare ltda , joinville , sc , brazil ) in contact with teeth exactly 6 hours per day for 8 consecutive days . no restrictions on dietary or hygiene habits were imposed , but the necessity to remove the residual solution after each whitening session was emphasised . every 48 hours , the volunteers were questioned about sensitivity of teeth or soft tissue . when hypersensitivity was confirmed , desensitising treatment was performed with 2% potassium nitrate ( desensibilize kf 2% , fgm dentscare ltda , joinville , sc , brazil ) . the four subjects were instructed to place the desensitizing gel in their tray and wear it for 20 minutes once a day , as recommended by the manufacturer . after detachment from teeth , the specimens were left to dehydrate for 96 hours before being gold sputter - coated to permit analysis in a scanning electron microscope ( dsm-940 a , carl zeiss , oberkochen , germany ) . digital sem photomicrographs were taken at four different areas over the surface of each fragment at 5000x magnification . enamel and resin surface alterations were classified qualitatively by increasing order of scores ranging from 0 ( no observable alterations ) to 4 ( heavy erosion with deep depressions ) . prior to blind analysis of the 160 images , a second assessment was repeated 4 weeks after the first evaluation , and the data obtained were considered as a whole for statistical comparison . statistical differences between the experimental and appropriate control groups were executed with the mann - whitney test adjusted to the 95% confidence interval . this study was initiated only after obtaining suitable approval from the local ethics committee , which is in conformity with the declaration of helsinki ( doh ) . all molars included fit the following criteria : 1 ) arising from different donors and 2 ) without structural damage , signs of hypomineralisation ( white spots ) or prior contact with saliva . after cleaning , teeth were individually positioned in a sectioning machine equipped with a water - cooled 300-m thick diamond saw ( minitorm , struers a / s , copenhagen , denmark ) . after that , the remaining crows were equidistantly sectioned in the mesiodistal and buccolingual directions to provide four enamel fragments ( 4 mm x 4 mm and 2 mm thickness ) each . the pieces obtained were moulded into a light regular - set silicon ( virtual light - body , ivoclair vivadent , so paulo , sp , brazil ) , and the moulds were filled with increments of a micro - hybrid composite resin ( ea2 opallis , fgm dentscare ltda , joinville , sc , brazil ) . moulds were sequentially polymerised over 20 seconds using a light - emitting diode unit set between 460 and 480 mw / cm ( ultra - blue i5 , dmc vasconcellos , so carlos , sp , brazil ) in order to produce enamel - like composite resin specimens . the resulting 40 specimens ( 20 of composite resin ) were then individually placed at the end of polyvinyl chloride cylinders ( 3/4 inch diameter and 20 mm high ) , which were filled with chemically - activated acrylic resin ( technovit 4000 , heraeus kulzer , wehrheim , germany ) . after 24 hours , the exposed side of each embedded fragment was flattened and polished by sequential use of wet 400- , 600- and 1200-grit silicon carbide papers ( norton,guarulhos , sp , brazil ) adapted in a slow - speed polishing machine set at 150 rpm ( ballerup , struers a / s , copenhagen , denmark ) . the finished specimens were then removed from blocks and ultrasonically cleaned for 5 minutes before being sterilised and stored in distilled water ( 370.5c ) until use . all volunteers presented the following inclusion criteria : absence of caries and gingivitis , no history of xerostomia , no previous bleaching therapy and willingness to understand and sign a consent form . participants had their maxillary right teeth cleaned with a rotary toothbrush and dentifrice before isolation with a rubber dam and stainless steel clamps . the buccal sides of the right upper second premolar and first molar were etched with 37% phosphoric acid ( dentsply , petrpolis , rj , brazil ) for 30 seconds and immediately washed with running water for 60 seconds . to fix the enamel and composite resin randomly on these surfaces , a two - step adhesive ( adper scotchbond , 3 m , sumar , sp , brazil ) was brushed and light - polymerised for 15 seconds before an increment of low - viscosity composite resin ( natural flow , dfl , jacarepagua , rj , brazil ) was used to glue the specimens on teeth using light - polymerisation for 40 seconds . these same procedures were performed on the adjacent lower right teeth to fix the enamel and composite resin on specimens not undergoing the bleaching process . an upper jaw impression was taken of each participant with alginate materials ( jeltrate , dentsply , petrpolis , rj , brazil ) , and impressions were poured with type ii dental stone ( pasom , so paulo , sp , brazil ) to fabricate a master cast . a 0.5 mm - thick wax layer was applied on the buccal face of each cast 1.5 mm away from the gingival margin to fabricate reservoirs for the bleaching gel . soft trays ( cristal , bio - art , so carlos , sp , brazil ) were made using a heat / vacuum tray - forming machine ( plasvac p7 , bio - art , so carlos , sp , brazil ) . the participants were instructed to use the tray at the upper arch and to keep the bleaching product ( whiteness perfect 16% , fgm dentscare ltda , joinville , sc , brazil ) in contact with teeth exactly 6 hours per day for 8 consecutive days . no restrictions on dietary or hygiene habits were imposed , but the necessity to remove the residual solution after each whitening session was emphasised . every 48 hours , the volunteers were questioned about sensitivity of teeth or soft tissue . when hypersensitivity was confirmed , desensitising treatment was performed with 2% potassium nitrate ( desensibilize kf 2% , fgm dentscare ltda , joinville , sc , brazil ) . the four subjects were instructed to place the desensitizing gel in their tray and wear it for 20 minutes once a day , as recommended by the manufacturer . after detachment from teeth , the specimens were left to dehydrate for 96 hours before being gold sputter - coated to permit analysis in a scanning electron microscope ( dsm-940 a , carl zeiss , oberkochen , germany ) . digital sem photomicrographs were taken at four different areas over the surface of each fragment at 5000x magnification . enamel and resin surface alterations were classified qualitatively by increasing order of scores ranging from 0 ( no observable alterations ) to 4 ( heavy erosion with deep depressions ) . prior to blind analysis of the 160 images , a single examiner was calibrated by viewing 30 additional sem micrographs . a second assessment was repeated 4 weeks after the first evaluation , and the data obtained were considered as a whole for statistical comparison . statistical differences between the experimental and appropriate control groups were executed with the mann - whitney test adjusted to the 95% confidence interval . representative sem images are shown in figures 14 , and the results of the mann - whitney test are presented in table 1 . following application of 16% carbamide peroxide , the enamel surface in the experimental group exhibited extensive mild erosion with shallow depressions and destruction of interprismatic matrix . this appearance differed significantly from that of non - bleached enamel fragments , which exhibited a smooth and amorphous aspect . the bleached composite resin specimens displayed a flat appearance , with slight erosion and some striation due to the grinding procedure . this appearance was statistically similar to that observed in the control group ( p<.05 ) . this study was the first to adhere human enamel and resin fragments to the buccal side of regular teeth to reproduce , as closely as possible , an actual home - bleaching situation . this manoeuvre permitted controlled and continuous exposure of specimens to saliva , beverages and oral hygiene habits , a procedure completely different from prior investigations in which removable appliances were adopted to carry the specimens.1,911 therefore , such biases make previous studies difficult to interpret and compare . scanning electron microscopy is a simple and effective method for identifying surface morphology alterations on enamel and composite resins submitted to home bleaching.1,12,13 nonetheless , dehydration and metal coating necessary to sem analysis are able to change part of the specimens structure;14 thus , the appearance of enamel observed may not correspond precisely to that of bleached enamel in its natural condition . porosity and erosion resulting from demineralisation were demonstrated by sem after in vitro and in vivo intermittent exposure of enamel to 10% carbamide peroxide.1,15,16 in the present study , the time of contact between the enamel and whitening gel ( 48 hours in total ) was shorter than those adopted in other investigations7,10,1517 and sufficient to produce an eroded appearance similar to that of acid - etched surfaces . this finding has already been observed in vitro by adebayo et al8 but not in vivo by metz et al,11 likely because the enamel specimens used in the latter research had prior contact with fluoride . the damage detected on enamel could be justified by the ability of urea , derived from the reaction of carbamide peroxide with water , to denature protein structures and thus cause structural and morphological changes through the degradation of organic molecules.14 in addition , the slightly acidic ph of the gel tested ( 6.04)18 may have contributed secondarily to these external modifications.12,17 however , a possible increased susceptibility of unerupted third molars enamel to the action of chemical agents such as carbamide peroxide should be considered before this result can be generalized . since these findings were detected in vivo , it may be suggested that the expected dissolving and buffering effects of saliva on the whitening gel19 or the attenuation provided for a peroxide - consuming enzyme on the surface of teeth20 were not able to minimise the deleterious impact of a highly concentrated bleaching gel on human enamel . alterations to enamel topography caused by low concentrations of bleaching gels may undergo repair over time by precipitation of mineral phases derived from saliva into the existing porosities.1,16,21 tooth remineralisation is a slow process , and the high concentration of bleaching gel used in this study probably smothered the saliva recovery properties . clinically , however , erosion reversal should be expected as soon as whitening gel application is discontinued . in recent laboratory investigations , the addition of fluoride to carbamide peroxide gel resulted in less demineralisation and shorter periods for enamel hardening recovery following bleaching.22,23 the absence of significant alterations on the surface of the composite resin bleached was already described for nano - hybrid and packable resins after extended periods of in situ and in vitro exposure to 15% carbamide peroxide.6,11 since morphological alterations were noticed on enamel specimens subjected to the same home - whitening conditions , we inferred that salivary attenuation of the highly concentrated peroxide carbamide substance did not completely respond for resin resistance to bleaching detrimental effects.19,20 enamel and composite are completely different materials , and an incompatibility between the solvents present in the whitening substances and the components in the polymer matrix of the resin - based material may justify the absence of deleterious effects on resin surfaces.2,11,13,24 another possible explanation for the absence of statistical difference between groups relates to the subjective and qualitative evaluations necessary for the sem micrographs and also to the low number of composite resin specimens used ; it is possible that major changes in the surface of bleached resin have been underrated , despite the effort to calibrate the examiners , thus reducing the numerical difference between groups . although not widespread as sem , atomic force microscopy ( afm ) enables quantitative and independent measurement of specimens surface morphology in their natural condition.17 thus , the possibility of getting different results , if afm had been used , can not be disregarded . colour improvements have been described separately for enamel25 and composite resin.26 since these structures were bleached together in this investigation , we provide evidence that these substrates responded differently to 16% carbamide peroxide . this phenomenon may justify the common observation of poor colour matching of pre - existing restorations after home whitening.11 nonetheless , given the diversity of composite resins available , the susceptibility of their surfaces to this or other vital bleaching regimens and formulations may be different . to date , it is uncertain how the surface changes that occur during bleaching are related to colour improvement and/or increases in staining susceptibility.5,6 however , the complete repolishing of aesthetically restored teeth to recover smoothness and delay staining of composite resins9,27 may both be unnecessary , at least for the material studied , and thus avoid the irreversible loss of enamel structure . within the limitations of this clinical study , we concluded that the occurrence of surface topography changes during home bleaching of aesthetically restored teeth with 16% carbamide peroxide was restricted to enamel .

objectives : this clinical study evaluated the effects of a highly concentrated home - bleaching agent on the surface morphology of aesthetically restored teeth.methods:specimens of human enamel and micro - hybrid composite resin were randomly adhered to the buccal side of right premolar and molar teeth of ten volunteers , who underwent a routine home - bleaching procedure with 16% carbamide peroxide on the upper jaw for 8 days . the mandibular unbleached specimens served as paired controls ( n=10 ) . ultra - structural assessment of the sample was carried out by scanning electron microscopy , and the resulting images were blindly evaluated for topographical alterations . the differences between groups were calculated with the mann - whitney test adjusted at the 95% confidence interval.results:the bleached enamel presented significantly more morphological changes than the control group . the aspect of resin composite exposed to the whitening substance was statistically similar to that observed in the corresponding control group ( p<.05).conclusions : the occurrence of surface topography changes during home bleaching of aesthetically restored teeth with 16% carbamide peroxide was restricted to enamel .

INTRODUCTION MATERIALS AND METHODS Preparation of specimens Bleaching process Surface analysis Statistical analysis RESULTS DISCUSSION CONCLUSIONS

PMC3034236

according to the conventional or folk psychological view , described above and illustrated in the left panel of figure 2 , a dog dreaming of chasing rabbits exhibits limb movements that resemble a dog running after its prey . as the romanes quote above attests , this view was in place long before active sleep was discovered in the middle of the last century ( aserinsky and kleitman , 1953 ) . soon after that discovery , it became clear that the cortex is not the driving force behind dreaming . rather , it is the brainstem that drives cortical activity during active sleep . it was this notion that helped inspire the activation - synthesis hypothesis of dreaming , which proposed that dreams arise from the synthesis by the cortex of internally generated signals arriving from the brainstem as well as previously stored information ( hobson and mccarley , 1977 ) . this was an elegant theory that directly and properly challenged the hegemony of the classic freudian interpretation of dreams . . left : the folk psychological view of dreaming in which dream activity directly stimulates some fully or partially realized form of motor activity . middle : discovering the importance of brainstem activity and inhibition of motor outflow during active sleep inspired a modified view of the relation between dreaming and twitching . now , the cortex is activated by the brainstem during active sleep to produce a dream , and the motor signals arising from this dream activity is largely suppressed . incomplete blockade of the motor outflow results in the production of twitching as a by - product of dream - related activity . ( 1966 ) , indicated in blue , posited a role for brainstem activity during active sleep in direct ascending activation of the cortex and descending activation of the musculature . this hypothesis is elaborated here to include an additional component , indicated in red , whereby brainstem - initiated twitching of the limbs generates sensory feedback , thereby activating the cortex . in turn , the hippocampus is activated as well ( not shown ) . by the time hobson and mccarley published their activation - synthesis hypothesis , it had already been discovered that active sleep is accompanied by a profound loss of muscle tone ( pompeiano , 1967a ) . this loss of tone is not merely the result of muscle relaxation ; rather , it results from active inhibition of the motor neurons that control the muscles . the quickly accepted resolution of this apparent paradox did not stray far from the folk psychological explanation of movements during sleep . as illustrated in the middle panel of figure 2 , the dreaming cortex was still viewed as producing movements , but now these movements were seen as mere by - products of dream activity bits and pieces of motor commands that are able to leak through an incomplete inhibitory filter . thus , when hobson and mccarley presented their model relating dream activity in the cortex with movements throughout the body , they explicitly endorsed the view of twitches as imperfectly realized manifestations of movements that would be expressed as organized movements but for the inhibition of muscle tone that accompanies active sleep . this fusing of folk psychology with sleep physiology seemed justified in light of the observation that adult cats with mesopontine lesions exhibit fully organized motor activity during active sleep as if the cats were acting out their dreams ( jouvet and delorme , 1965 ) . hobson and mccarley ( 1977 ) described this curious phenomenon like this : the animals show all of the major manifestations of [ active ] sleep except the atonia ; instead of the fine twitches of the digits and the limb jerks that are normally present in [ active sleep ] , these cats display complex motor behaviors including well - coordinated attack and defense sequences that have no apparent relationship to the environment . ( p. 1337 ) . brief episodes of an otherwise integrated behavior that is suppressed by the presence of motor inhibition ( p. 568 sleep can continue undisturbed . and as for the small twitches that break through the inhibition , they are apparently without purpose ( p. 566 ) . if twitches were mere remnants of integrated behavior descending from the cortex , then we would expect that disconnecting the cortex from the brainstem would eradicate twitching . but this is not what was found . to show this , experimenters completely transected the brain into two parts . ( this method has long provided valuable insights into the neural circuitry governing sleep wake processes , beginning with bremer 's ( 1935 ) cerveau isol preparation . ) for example , when villablanca ( 1966 ) performed transections in adult cats that prevented cortical communication with the brainstem , he reported periods of active sleep , including muscle twitches , that could not be distinguished from similar periods in non - transected cats . similarly , using 1-week - old rats , we performed transections that passed through or anterior to the region within the brainstem that has been implicated in the production of active sleep in adults ( kreider and blumberg , 2000 ) . however , when transections were performed more rostrally such that they left the brainstem region intact but still completely disconnected the brainstem from the forebrain , the quantity and quality of twitching were completely unaffected . subsequently , we identified neurons within the laterodorsal tegmental nucleus ( ldt ; a nucleus that was spared by the rostral transections discussed above ) that fire in close temporal proximity to limb twitching ( karlsson et al . , 2005 ) ; moreover , when electrolytic lesions were produced that included the ldt , the amount of twitching was significantly reduced despite increased amounts of sleep . all together , these findings demonstrate that twitching , in infants and adults , is not the product of dreamwork descending from the cortex . in addition to twitching , villablanca ( 1966 ) reported that rems in his adult cats were unaffected by the same transections that spared limb twitching . for this and other reasons , it is worth considering the similarities between rems and twitching . in their paper first reporting the discovery of rem sleep , aserinsky and kleitman ( 1953 ) immediately floated the possibility that rems reflect the visual content of dreams , as if subjects watch their dreams . indeed , the scanning hypothesis has itself become a part of the folk psychological interpretation of dreaming . although the scanning hypothesis remains popular , rems may not provide the insight into dreaming that many have assumed . this is the view of chase and morales ( 1990 ) , who hypothesized that the rapid eye movements themselves can be viewed as myoclonic activity ( p. 560 ) . according to this view , eyes are just another kind of limb and are , like the other limbs of the body , controlled by skeletal muscles that twitch during active sleep . we tested this hypothesis in infant rats by recording from the eye muscles at ages before and after the emergence of sleep - related eye movements ( seelke et al . , 2005 ) . consistent with the chase and morales hypothesis , the eye muscles conformed to expectations for any skeletal muscle : the tone of the eye muscles increased during waking and decreased during sleep , and twitching of the eye muscles was detected at all ages examined even at ages when the eyelids are sealed and the eyes do not freely move ( in rats , the eyelids do not open until 15 days of age ) . thus , although the eyes have occupied a special position in the sleep literature , it may be that they are no more special than any other limb of the body . if so , then anything that we discover about the functional importance of limb twitching for the developing infant may apply with equal relevance to rems and vice versa . howard roffwarg was a young psychiatric resident in the early 1960s when he first began recording brain activity in young infants . working with the pioneering sleep researcher william dement , roffwarg predicted that active sleep would not be detectible until 2 years of age when children are first able to talk about their dreams such was the strong link that had already been forged between active sleep and dreaming . so they were surprised to discover that not only is active sleep detectible at birth , but that the quantities of active sleep are highest at birth and even higher in preterm infants . the writing of the paper reporting these results took several years ; by then they were convinced that any hypothesis which purports to account for the regulation of rem sleep will eventually have to explain the great quantities of rem sleep during early development ( roffwarg et al . , 1966 , in their paper , roffwarg and colleagues turned away from dreaming to explain the great quantities of newborn active sleep . although work over the previous decade had established a strong association between rems and dreaming , they noted that rems persist even in the congenitally blind as well as in humans , cats , and kittens without a functioning cortex . so they looked beyond dreaming to the brainstem activity that drives active sleep , including the forebrain activation and motor events that comprise it ( see figure 2 , right panel , blue boxes and arrows ) . because active sleep is so prominent in the newborn period and declines with age , and because the newborn 's waking life is limited in time and scope and offers little occasion for stimulation ( p. 614 ) , they hypothesized that the vigorous neural stimulation arising from the brainstem during active sleep substitutes for the lack of waking stimulation . specifically , they suggested that this sleep - related stimulation assists in a variety of developmental processes , including neuronal differentiation , maturation , and myelinization in higher centers ( p. 616 ) . as for the eventual emergence of dream imagery in children , they expressed a view that presaged the activation - synthesis hypothesis , which would not be formalized for another 10 years : perhaps , they wondered , ; if so , the dream would truly appear to be born in the brainstem but clothed in the cortex the first empirical tests of the ontogenetic hypothesis , which were not performed until the 1980s , focused on the role of brainstem - generated ponto - geniculo - occipital ( pgo ) waves in the development of the visual system in kittens . consistent with the ontogenetic hypothesis , pgo waves appeared to contribute to the development and neural differentiation of the lateral geniculate nucleus , a nucleus that receives visual information from the retina and transmits it to visual cortex ( davenne and adrien , 1984 , 1987 ; davenne et al . subsequent work from roffwarg 's laboratory indicates that active sleep modulates visual cortical plasticity early in development ( shaffery et al . significantly , these tests of the ontogenetic hypothesis have two important features in common : they focus on direct stimulation of the forebrain by the brainstem ( in the form of pgo waves ) and they relate exclusively to the visual system . there is no strong rationale for limiting the developmental effects of active sleep on forebrain development to either the visual system or to direct stimulation from the brainstem . here i aim to make room within that hypothesis for twitch - related sensory feedback as a source of indirect stimulation of the forebrain . but to provide a more prominent place for twitching within the ontogenetic hypothesis and its central theme of activity - dependent brain development , we must demonstrate that sensory feedback arising from twitching limbs actually modifies brain activity . here we immediately face an obvious problem : namely , based on decades of research , sensory inputs appear to be dampened during sleep , especially during active sleep ( pompeiano , 1967b ; taepavarapruk et al . , 2002 ) . the phenomenon of increased sensory threshold during sleep is widespread : it has been shown using auditory ( baust et al . , 1964 ) and olfactory ( badia et al . , 1990 ) stimuli in adult humans , auditory ( hutt et al . , 1968 ) and olfactory ( murray and campbell , 1970 ) stimuli in human neonates , and even vibratory stimuli in flies ( shaw et al . , 2000 we also reported increases in olfactory threshold during sleep in week - old rats ( seelke and blumberg , 2004 ) . with so much evidence consistently pointing to a diminution of sensory experience during sleep , it is perhaps unsurprising that the folk psychological interpretation of twitching as a by - product of dreaming would not have been questioned : animals may twitch all they want , but inhibition of sensory input would prevent the brain from receiving sensory information from a twitch . until recently , there was no reason to doubt the assumption that any sensory feedback arising from twitching would be blocked or substantially diminished from entering the nervous system . the first strong evidence in favor of a functional role for twitches during sleep in infant rats came from jens schouenborg 's group in sweden ( petersson et al . , , they showed that tactile feedback , produced when a twitching limb touches a nearby object , guides the organization of spinal sensorimotor circuits . thus , at least at the level of the spinal cord , sensory input provided by twitching is not blocked . the next breakthrough came when investigators recorded from somatosensory cortex during periods of twitching in infant rats ( khazipov et al . , 2004 ) . in light of previous work that seemed to show that cortical activity was barely detectible at these early ages , the findings reported by khazipov and colleagues were surprising . what they discovered was a new cortical event they called it a spindle burst comprising a brief wavelike burst of activity ( a typical spindle burst might oscillate at 15 hz for 1 s ) . even more surprising was that these spindle bursts were triggered by twitches during sleep in a topographic manner : forelimb twitches triggered spindle bursts in the forelimb region of somatosensory cortex ; hindlimb twitches triggered spindle bursts in the hindlimb region , etc . that the causal arrow flowed from limb twitch to cortex was established by transecting the spinal cord , thereby blocking the flow of sensory information from the hindlimbs , and effectively decreasing the number of spindle bursts produced . subsequent work from this group demonstrated that spindle bursts also occur in visual cortex in response to retinal waves ( hanganu et al . , 2006 ) and in barrel cortex in response to whisker movements ( minlebaev et al . , 2007 ) . thus , this newly discovered cortical event perhaps the most prominent cortical event in the developing cortex may play a critical role in organizing cortical circuits involved in the processing of sensory information . we further explored the relationship between spindle bursts and sensory processing using an experimental paradigm that allowed us to observe twitching and also deliver discrete sensory stimulation to the forepaw ( marcano - reik and blumberg , 2008 ) . in these experiments , we simultaneously recorded from the region of somatosensory cortex that processes information from the forepaw . first , we found that spindle bursts could be reliably evoked by forepaw stimulation during sleep or during wakefulness . second , we found that stimulating the forepaw in such a way as to produce tactile stimulation of the plantar surface and dorsiflexion of the wrist that is , touching and flexing produced a spindle burst that was superimposed within a second cortical event that we called a slow potential . we were able to dissociate the two events by stroking the plantar surface alone thereby producing only the slow potential or by flexing the wrist alone thereby producing only the spindle burst . accordingly , we suggested that limb movements during twitching produce proprioceptive feedback that triggers spindle bursts ; in contrast , the slow potential would only be produced when a twitching limb makes contact with another object ( e.g. , a littermate or mother ) . although we now know that twitching in early development drives cortical activity , we do not yet have a sense of the magnitude of this activity . in another developmental domain the emergence of locomotion in human infants a compelling argument has been made about the formative role of experience by simply documenting the sheer quantity of locomotor activity that infant crawlers and walkers exhibit ( adolph , 2005 ) . for example , each day an infant crawler covers , in short bursts , the lengths of two football fields . in light of such evidence , the formative role of locomotor experience is difficult to ignore . we can take a similar approach with twitching . we routinely record activity in the nuchal muscle the muscle located at the back of the neck that is responsible for holding our head upright . from these recordings we can count the number of twitches that occur . when we did this , we estimated that this one muscle twitches over 38,000 times each day ( marcano - reik et al . , 2010 ) . because we were recording cortical activity in these same animals , we were able to similarly quantify the number of spindle bursts : we estimated that within one discrete region of somatosensory cortex , 4,000 spindle bursts are generated each day . as shown in the right panel of figure 2 and elaborated in figure 3 , a fresh perspective has emerged that departs substantially from the dominant view that has guided our thinking about twitching until now . with the addition of sensory feedback , twitching is potentially transformed from a functionless by - product of dreaming to an active contributor to sensorimotor integration within the spinal cord and brain , including the development , refinement , and maintenance of somatotopic maps . indeed , it may be that twitching contributes to the functional linking of muscle and somatosensory cortex in a way that is similar to how retinal waves contribute to the linking of retina and visual cortex ( katz and shatz , 1996 ; wong , 1999 ) . this notion of activity - dependent development was at the heart of roffwarg et al.s ontogenetic hypothesis , and so the view of twitching promoted here should be considered an elaboration of that hypothesis . the likely pathway from the triggering of a twitch to the processing of twitch - related sensory feedback in the forebrain . neurons within the brainstem trigger a limb twitch , whereupon sensory feedback , including proprioceptive feedback , is generated . proprioceptive feedback is communicated through the spinal cord , dorsal column nuclei , and thalamus before generating a spindle burst in primary somatosensory cortex . from the cortex in addition , interactions between homotopic regions of left and right somatosensory cortex , via the corpus callosum , modulate spindle burst activity . in support of a functional role for twitching , we were recently able to document a relationship among twitching , spindle burst production , and the plasticity of somatosensory cortex ( marcano - reik et al . , in press ) . to demonstrate a change in plasticity , we relied on a previous finding that transecting the corpus callosum the large bundle of fibers that connects the two cerebral hemispheres immediately decreases the reliability with which spindle bursts occur in response to forepaw stimulation , from nearly 100% to 70% ( marcano - reik and blumberg , 2008 ) . in our subsequent study , we again transected the corpus callosum , but now we assessed recovery of function over the ensuing week . we found that recovery of function occurred only if the corpus callosum was transected at or before 6 days of age . after that age , recovery of function did not occur ( in fact , the reliability of responding worsened ) , thus indicating a reduction in cortical plasticity . also around 6 days of age , we noted several other dramatic developmental changes , including an abrupt transition in how the corpus callosum modulates spindle bursts , a rapid increase in intrinsic cortical synaptic connectivity , and a transformation in gaba 's functional properties . that these changes occur contemporaneously with an abrupt decrease in the rates of twitching provides further support for the hypothesis that this sleep - related behavior participates in the activity - dependent development and refinement of cortical networks . the functional consequences of twitching for the developing brain may not end with the cortex . in fact , the hippocampus also exhibits activity patterns that are strongly linked with twitching ( mohns and blumberg , 2008 ) . recording from the hippocampus of 1- to 12-day - old rats , we found that the majority of hippocampal neurons increase their firing rates during periods of myoclonic twitching of the limbs , and a subset of these neurons exhibit a burst of activity immediately after a limb twitch , again suggesting that the twitches themselves provide sensory feedback to the infant hippocampus . we next showed that disconnecting the cortex and hippocampus had no effect on the production of spindle bursts but eliminated twitch - related activity in the hippocampus , thus revealing the path of activation from cortex to hippocampus ( mohns and blumberg , 2010 ) . we do not yet know whether twitch - related activation of the brain continues beyond the hippocampus . but for now , we propose that twitching not only contributes to the development and refinement of neural circuits within cortex and hippocampus , but also helps coordinate interactions between those two structures , whose integrated relations are critical for learning and memory . how can we be sure that twitching per se is important for central nervous system development ? after all , cortical spindle bursts occur during waking movements such as kicking and stretching . at this time it is a fact of mammalian development that twitching is associated with active sleep and active sleep occupies the plurality of time of the young infant . by contrast , early in development , wakefulness and its associated behaviors are relatively rare . ( 1966 ) hypothesized that active sleep can be interpreted as a means of stimulating the brain in the absence of waking stimulation . according to this view , active sleep provides a substitute for the stimulation that wakefulness will provide later in life . the notion that stimulation of any kind during sleep or waking may be sufficient to drive activity - dependent processes raises the key distinction between permissive and instructive roles of activity in brain development ( crair , 1999 ) . when neural activity has permissive effects , it only matters that some form of activity occurs . accordingly , if twitching played a permissive role in brain development , the temporal and spatial qualities of a twitch or the fact that it happens during active sleep would not matter . in this broad sense of permissive , all that would be needed for the development of sensorimotor integration is some form of activation , regardless of its source or patterning . if so , then perhaps twitching is favored over waking movements only because twitching stimulates the brain without arousing the infant . in the visual system , long before animals experience patterned visual input , rhythmic bursting activity within each retina contributes to the development and refinement of connections among retina , thalamus , and visual cortex ( katz and shatz , 1996 ; wong , 1999 ) . because the source and patterning of these retinal waves influence the kinds of downstream effects observed , they are viewed as playing an instructive role in visual system development ( crair , 1999 ) . the question we face is whether twitching plays such an instructive role in the development of the sensorimotor system . there are several features of twitching that may contribute to its playing an instructive role . first , if twitching were to help establish precise somatotopic relations between muscle and brain , then it would be important that each twitch movement be discrete . we have shown that twitch movements in rat fetuses and newborns are discrete , rarely if ever occurring simultaneously with twitches in other limbs ( robinson et al . , 2000 ) . in contrast , waking movements including stretching and kicking are not only larger movements involving many muscle groups within a limb , but they also often involve simultaneous movements in more than one limb . thus , just as retinal waves contribute to the segregation of inputs from the two eyes , twitches may help to segregate inputs from separate muscle groups within a limb and between different limbs . second , twitches occur during active sleep when muscle tone is low . as a consequence , sensory feedback from small movements will be easier to detect during active sleep ( i.e. , the signal - to - noise ratio will be higher ) . when muscle tone increases during wakefulness , background noise will also increase , thereby obscuring sensory feedback arising from relatively small movements . thus , the temporal patterning of twitching and its occurrence during periods of low muscle tone may work together to ensure that the nervous system establishes clearer associations between motor outputs and sensory inputs . if so , then twitching may promote neural development in part because of its association with active sleep . for example , acetylcholine ( ach ) release from the basal forebrain of adults is highest during active sleep ( vazquez and baghdoyan , 2001 ) and ach is an established modulator of cortical plasticity ( bear and singer , 1986 ; rasmusson and dykes , 1988 ; tremblay et al . , 1990 ; juliano et al . , thus , it is possible that active sleep , through release of cortical ach , sets the stage for twitching to modify somatosensory circuits . efference copy is a well - established mechanism by which animals distinguish self - produced movements from sensations from movements evoked by exogenous stimuli ( blakemore et al . , 2000 ; cullen , 2004 ) . accordingly , in addition to the caudally projecting signal that results in a twitch , there may be a rostrally projecting signal that prepares the nervous system for the ensuing arrival of twitch - related sensory information , thereby contributing to sensorimotor development and somatotopic organization . this efference copy could be transmitted to the cholinergic basal forebrain , which could then modulate somatosensory cortex in anticipation of the ensuing arrival of sensory information . indeed , in infant rats , ach released from the basal forebrain contributes to spindle burst production in visual cortex ( hanganu et al . , 2007 ) . the historical progression of ideas illustrated in figure 2 may also help us make sense of other sleep - related phenomena . for example , rem behavior disorder ( rbd ) is a degenerative disease that affects mostly older men and has a prevalence of approximately 0.5% ( fantini et al . , 2005 ) . as with the cats with mesopontine lesions described earlier , individuals with rbd enter active sleep but , instead of exhibiting the normal features of muscle atonia and twitching , they behave in ways that suggest they are acting out their dreams these behaviors are many and varied : they include yelling , grabbing , punching , and kicking . subsequent analyses of cats with mesopontine lesions called into question the enticing idea that they were acting out their dreams ( morrison , 1983 ) . instead , it became clear that these lesions were not only preventing muscle atonia , they were also disinhibiting locomotor drive . but if these cats were not acting out their dreams , how might we account for the relation between the reported dreams of rbd patients and their dream - enactment behaviors ? the conventional interpretation of rbd seems to fit well with the scheme presented in the middle panel of figure 2 : brainstem activity drives cortical activity , dreams are produced , and the absence of motor inhibition in rbd allows the motor features of dreams to be implemented without interference . what if the right panel of figure 2 comes closer to explaining the dream behavior relationship in rbd ? that is , given that brainstem activity during active sleep drives motor activity , the damaged brainstems of rbd patients may result in the production of more robust sleep - related behaviors . then , the sensory feedback from these vigorous movements could trigger the violent dreams of rbd . ( 2005 ) suggest that the increase in sleep - related motor activity in rbd may be responsible for both motor behaviors and action - filled dreams ( p. 1014 ) . behavior causal sequence in rbd as perfectly consonant with the activation - synthesis hypothesis of dreaming . i agree , although it should be noted that hobson and mccarley ( 1977 ) do not appear to have envisioned a role for sensory feedback in the modulation of dream content . still , just as the ontogenetic hypothesis can incorporate sensory feedback from twitching while still retaining its central theme of activity - dependent development during sleep , the activation - synthesis model can incorporate an unanticipated form of cortical activation . indeed , given that sleepers often incorporate sensory information into dreams ( dement and wolpert , 1958 ; berger , 1963 ) , can we imagine that patients with rbd would fail to incorporate their own yelling , grabbing , punching , and kicking into their dreams ? each infant faces the challenge of adapting and functioning in the moment even as its body grows , limbs elongate , muscles strengthen , brain differentiates and forms new connections , and ecological niches and social interactions change ( alberts and cramer , 1988 ) . preprogrammed and prescribed solutions will not do because the problems to be solved around the next bend can not be foreseen . this principle applies to all of us , whether typically or atypically formed ( blumberg , 2009 ) . it is in this context that we may be able to make sense of the great quantities of sleep in newborns . although twitching has long been viewed as by - products of a dreaming brain , we now have ample reason to move on to new ideas . of course , we might have questioned the by - product hypothesis on the simple grounds that so much twitching in a developing infant wastes energy that would be more wisely devoted to growth . but that argument alone could never have displaced the supremacy of the by - product hypothesis . that spontaneous activity plays a critical role in the development of the nervous system is now a foundational concept in the field ( shatz , 1990 ; purves , 1994 ; o'donovan , 1999 ) . and can there be any more conspicuous and dramatic example of spontaneous activity than twitching in newborns ? of course , it is possible that all this twitching might have had no effect on neural activity . indeed , this possibility was assumed to be true because of the well - known increase in sensory thresholds that accompanies sleep . however , it is now clear that sleep - related increases in sensory threshold do not prevent the feedback from these self - generated movements from entering the central nervous system . on the contrary , sensory feedback from twitching thus , we are now able to glimpse like never before how infant sleep may contribute to developmental flexibility and evolutionary change ( blumberg and seelke , 2010 ) . it is important to determine whether twitching plays a permissive or instructive role in development . in addition to its effects on neural development , twitch movements may influence the development of bone , cartilage , and muscle ( blumberg and lucas , 1996 ; mller , 2003 ) . also , although twitching does not end in infancy , we do not yet have a clear picture of the quality and quantity of twitching across the lifespan or in different species , including humans . we also do not know if the adolescent or adult brain experiences the sensory feedback from twitching as infants do . this new view of twitching should open new and potentially fruitful avenues for basic research and clinical application . for example , twitches may prove to be sensitive and powerful tools for plotting the trajectory of normal and abnormal somatosensory development across the lifespan ; for diagnosing sensorimotor dysfunction and recovery of function after peripheral injury and brain damage ; and for assessing the effectiveness of therapies designed to improve sensorimotor function after injury . perhaps most intriguing is the possibility that the contributions of twitching to somatosensory plasticity in normal development are recapitulated during recovery from stroke or brain injury , or are recruited as amputees learn to rely more heavily on their remaining limbs . important clues to the relations between sleep - related motor activity and neuropathology are already emerging . consider once again rbd , a neurodegenerative disorder characterized by disruption of the brainstem mechanisms that normally inhibit motor activity during active sleep . interestingly , clinical investigators have been developing increasingly precise and quantitative measures of twitching with the hope that such measures will facilitate improved understanding of the neurological deficits in rbd ( lapierre and montplaisir , 1992 ; bliwise et al . , 2006 ; frauscher et al . , 2008 ; iranzo et al . , for example , although investigators typically emphasize the dream - enactment aspects of rbd , twitching in these patients increases during active sleep even on nights when overt behavioral abnormalities are not expressed ( bliwise and rye , 2008 ) . as surprising as rbd is , even more surprising is that rbd patients are at much greater risk of developing parkinson 's disease and other related disorders ( schenck et al . , 1996 ) . in fact , rbd can precede the onset of these other disorders by many decades ( claassen et al . , 2010 ) . in other words , a dysfunction of motor activity and muscle control during active sleep has now been associated with a devastating degenerative motor disease of old age . moreover , this association is made even stronger by observations , in parkinson 's patients , of increased twitching during sleep ( bliwise et al . , 2010 ) and even evidence of restored motor control during sleep ( de cock et al . , 2007 ) . such observations suggest that systematic and precise measures of twitching in humans could prove useful for the diagnosis , treatment , and , perhaps someday , prevention of these and other neuropathological conditions . it is difficult to foresee all possible directions for this research because so little is known about twitching across the lifespan in health and disease . . then we will be in a better position to generate hypotheses and test them in humans and other animals . ultimately , by seriously considering twitching as a complex behavior in its own right , we may someday come to appreciate that these seemingly inconsequential movements are more meaningful than anyone ever dreamed . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

conventional wisdom has long held that the twitches of sleeping infants and adults are by - products of a dreaming brain . with the discovery of active ( or rem ) sleep in the 1950s and the recognition soon thereafter that active sleep is characterized by inhibition of motor outflow , researchers elaborated on conventional wisdom and concluded that sleep - related twitches are epiphenomena that result from incomplete blockade of dream - related cortical activity . this view persists despite the fact that twitching is unaffected in infants and adults when the cortex is disconnected from the brainstem . in 1966 , roffwarg and colleagues introduced the ontogenetic hypothesis , which addressed the preponderance of active sleep in early infancy . this hypothesis posited that the brainstem mechanisms that produce active sleep provide direct ascending stimulation to the forebrain and descending stimulation to the musculature , thereby promoting brain and neuromuscular development . however , this hypothesis and the subsequent work that tested it did not directly address the developmental significance of twitching or sensory feedback as a contributor to activity - dependent development . here i review recent findings that have inspired an elaboration of the ontogenetic hypothesis . specifically , in addition to direct brainstem activation of cortex during active sleep , sensory feedback arising from limb twitches produces discrete and substantial activation of somatosensory cortex and , beyond that , of hippocampus . delineating how twitching during active sleep contributes to the establishment , refinement , and maintenance of neural circuits may aid our understanding of the early developmental events that make sensorimotor integration possible . in addition , twitches may prove to be sensitive and powerful tools for assessing somatosensory function in humans across the lifespan as well as functional recovery in individuals with injuries or conditions that affect sensorimotor function .

Chasing Rabbits The Ontogenetic Hypothesis The Ontogenetic Hypothesis Elaborated Is Twitching Permissive or Instructive? Rem Behavior Disorder (RBD) and the Activation-Synthesis Hypothesis Revisited Conclusions and Future Directions Conflict of Interest Statement

PMC3944307

cerebral palsy ( cp ) describes a group of permanent disorder of the development of movement and posture causing activity limitation , that are attributed to non - progressive disturbances that occurring in the developing fetal or infant brain . the motor disorders of cerebral palsy are often accompanied by disturbances of sensation , perception , cognition , communication , behavior , epilepsy , and secondary musculoskeletal problems1 . cp is accompanied by a wide range of problems and has a broad spectrum of symptoms , making evaluation of the patient and setting of rehabilitation goals difficult . in the evaluation of a child with cp , the type of involvement is identified , the functional condition and secondary deformities are assessed , the needs of the patient are identified and a rehabilitation program is designed2 . rehabilitation programs for children with cp should be appropriate for the age and functional condition of the patients . the aim of cp rehabilitation should be to minimize disability and to promote independence and social participation2 . although the concept of cp rehabilitation has changed in recent years to focus on patient participation in everyday activities , the neurophysiological approach is still used within the combined therapy methods . the neurophysiological approach is a specific strategy based on the fact that sensory stimuli sent by various methods cause reflex motor responses . facilitation or inhibition of muscle groups via the stimulation of exteroreceptors and proprioceptors is the aim of this approach3 . the bobath technique is the most common method of motor stimulation and it is used worldwide . in this technique , a child is positioned in reflex - inhibiting postures ( rip ) to reduce spasticity . therapists stimulate key control points in the body , triggering reflexes that provide head and body control2 . the primary aim of the present study was to evaluate the results of bobath - based rehabilitation for pediatric cp inpatients . the study subjects were 28 children with cp who were inpatients at a pediatric rehabilitation inpatient clinic . inclusion criteria were : being an inpatient of our hospital aged 212 with a diagnosis of cp ; having one permanent primary caregiver ; and the caregiver having no medical , chronic or psychotic problem . a cp evaluation form was completed during detailed locomotor system , neurologic and orthopedic examinations prior to the rehabilitation program . the gross motor function measure ( gmfm ) the patients were categorized according to gross motor function classification system ( gmfcs ) . gmfcs is a standard 5-level system used to classify the gross motor functions of children with cp , which was developed by palisano et al . in 19974 . gmfm is a standardized observational test used to measure temporal changes in gross motor functions of children with cp . a manual and cd of the criteria were prepared , with the approval of russel et al . , and were bought to the study sessions , and patients were scored according to this manual6 . all of the patients received bobath treatment , administered by a physiotherapist , for 1 hour per day , 5 days a week . furthermore , during the hospitalization period the patients permanent caregivers were informed about cp and were given instruction in a home exercise program . the locomotor system , neurologic and orthopedic examination , gmfm , and gmfcs evaluations , were evaluated at the time of admission to hospital , and again at discharge from hospital , and at 1 and 3 months after discharge . the duration of hospitalization was determined by a physiatrist supervisor who was unaware of which patients were included in the study . the examination used evaluation methods such as visual acuity , eye movements , vep ( visual evoked potential ) , and erg ( electroretinography ) . children in the 412 age group were evaluated using the sd porteus and kent egy tests by a psychologist specializing in related tests . the time of discharge was decided by a clinical trainer specialist who was unaware of which children were included in the study . since the patients were of pediatric age , written consent for participation was obtained from their parents or legal guardians . the wilcoxon paired test and paired t - test were used after repeated measures anova , to compare patient data recorded at different times . spearman s correlation test was used to assess the correlation between cp type and mental condition . however , 15 patients were excluded from the study and the results of only the remaining 28 patients were evaluated . the reasons for exclusion from the study included patients desire for discharge before the completion of rehabilitation aims , 2 patients , and irregular or non - attendance at follows up for the remainder . the 28 children with cp who were evaluated were within the 212 age group ( mean 6.962.82 years ) , 12 ( 42.9% ) were male , and 16 ( 57.1% ) were female . twenty - four of the patients were spastic in terms of clinical type ( tables 1 and 2table 1 . classification of our cp patients ( according to european cp follow - up group)1sp type ( new classification)n%spastic bilateral2071.43spastic unilateral414.29dyskinetic27.14mixed27.1428100table family histories showed that the parents of 10 ( 35.7% ) of the participants were first - degree relatives . average age of diagnosis was 11.914.19 months ; average age until rehabilitation was 35.630.56 months . the average interval from time of diagnosis to start of rehabilitation was 23.729.88 months . although 71.4% of the children were identified with a problem within their first two years , only 9 ( 32.1% ) started rehabilitation ( table 3table 3 . identification of the problem and rehabilitation starting time in cp patientsn%identification of the problem before 12 months of age2071.4identification of the problem after 12 months of age828.6starting rehabilitation before 12 months of age932.14starting rehabilitation after 12 months of age1967.86 ) . of the 28 patients , these were the most frequent findings , followed by strabismus and optic disc paleness which is a retinal examination finding ( table 4table 4 . diagnosis of cp patients after eye examinationdiagnosissub - group diagnosisnrefraction defecthypermetropia14myopia4astigmatism12strabismusesotropia4exotropia3pale optic disc6otheroptic nerve coloboma1traumatic optical atrophy1 ) . analysis of speech problems showed that 6 ( 21.43% ) patients had normal speech , while 7 ( 25% ) had dysarthria , 9 ( 32.4% ) were able to speak 12 words and 6 ( 21.43% ) could not speak . three ( 15% ) were found to have normal intelligence ; 7 ( 35% ) had mild mental retardation ; 8 ( 40% ) had moderate mental retardation , and 2 ( 10% ) had severe mental retardation . we found no correlation between the degree of mental retardation and cp type ( r=0.11 p=0.932 ) . level 5 included 9 patients at the time of admission and 6 at the 3-month follow - up . similarly , the number of patients in level 4 decreased from 11 at the time of admission to 9 at the 3-month follow - up . conversely , the number of patients in level 3 increased from 2 to 7 . there was no significant difference in the gmfm total score between 1 and 3-month follow - up . however , in a comparison of pre - hospitalization score with post - discharge scores at 1 and 3 month follow - up , both the post - discharge scores showed significant increases when comparing with admission . however there was a significant decrease in gmfm score between discharge and 1st month ( table 5table 5 . comparison of gmfm scoresgmfm total scoremeansdp valueadmission34.0228.950.002*discharge41.0828.55admission34.0228.950.001 * 1 month follow - up39.6226.50admission34.0228.950.001 * 3 month follow - up40.3527.02discharge41.0828.550.006 * 1 month follow - up39.6226.50discharge41.0828.550.007 * 3 month follow - up40.3527.021 month follow - up39.6226.500.0963 month follow - up40.3527.02*p<0.01 ) . comparison among the four evaluation times showed a significant increase in sitting and standing parameters at 1-month and 3-month follow - up ( p<0.05 ) . crawling and kneeling sub - groups only showed a significant decrease between discharge and 1-month follow - up scores . similarly , walking running jumping scores showed a significant increase at the time of discharge , a decrease at 1-month follow - up and an increase at 3 month follow - up ( p<0.05 ) of the permanent caregivers , 24 ( 85.71% ) were the patient s mother , while the caregivers in 4 cases ( 14.29% ) were other relatives ( aunt , grandmother , stepmother ) . cp has a mean incidence of 23/1000 , although it shows variations on a country basis7 . in a multi - centric cross - sectional study carried out in 27 cities in 1996 including 146 doctors 50,000 children aged 016 , the prevalence of cp was 0.2% in turkey8 . since the presence and severity of accompanying problems shows wide variation , cp is regarded as a group of symptoms rather than a disease . therefore , rehabilitation of individuals with cp requires a multidisciplinary approach that addresses the patients needs more than the disease . evaluation of cp rehabilitation results are rather difficult , since patients have differing developmental and motor levels . it is therefore difficult to determine whether observed improvements are the result of rehabilitation or a natural outcome of growth and development . the use of 1-month and 3-month follows- ups permitted ongoing monitoring of rehabilitation and developmental outcomes at home following discharge from the hospital . thus , the information provided to caregivers about their children s condition was repeated , and they were encouraged to perform certain exercises at home . however , a large proportion of patients failed to attend the follow - ups , and were lost to follow - up . in the present study , this differs from a study carried out in 14 centers in europe , which reported that the proportion of male patients was higher ( m / f=1.33)9 . the spastic type of cp is most prevalent1 , comprising approximately 75% of all cases7 . similarly , in this study , the spastic type had the highest prevalence . early diagnosis of cp is important for the start of early rehabilitation . the caregivers were asked to report the first identification of the child s problem . although 71.4% of the patients were identified with problems in the first year of life , only 31.4% started rehabilitation in the first year . , the level of diagnosis before the age of 2 was 35% , while 87% of patients were diagnosed before the age of 511 . among our patients , similarly , 7 patients had strabismus , and 6 patients had pale optic disc , which is a retinal examination finding . in a study by yksel et al . they found that 43.9% of patients had strabismus , while 24.3% had refraction defects12 . similarly , in a meta - analysis , ashwal et al . reported that 28% of cp patients had visual impairment and ocular motility13 . in a multicentric study carried out in europe , 11.1% of the patients with cp the differing prevalence of visual impairment conditions is associated with variations in the time and severity of brain damage , involvement location , and thus the occurrence of different visual problems according to cp type . the literature contains studies to define visual findings in a cp type14 , 15 . however , the important thing is that children with cp commonly experience visual problems ; therefore , eye examination should certainly be made , even if there is no visible finding . the incidence of mental retardation was reported as 3050%16 , 17 . in our study , 3 ( 15% ) patients had normal intelligence , 7 ( 35% ) had mild mental retardation , 8 ( 40% ) had moderate mental retardation and 2 ( 10% ) had severe mental retardation . approximately one - third of those with mental retardation had a mild degree of retardation . athetotic types have better mental conditions than others . on the other hand , severe mental retardation is observed in spastic quadriplegic children with rigid , atonic and severe involvement16,17,18,19 . in this study , there was no correlation between the degree of mental retardation and cerebral palsy type . another significance of mental condition in children with cp is that , as the degree of mental retardation increases , life expectancy decreases18 . gmfm is a motor function criteria designed by russel et al.20 to measure the effectiveness of physical therapy in cp patients . in a study of 111 children with cp , evaluation scores of physiotherapists , families and blind evaluators were found to be significantly correlated . nordmark et al . reported that gmfm provided reliable scoring between different evaluators and scoring at different times by the same evaluators21 . gmfm is a valid and reliable method that has been commonly used in recent years to evaluate the effects of physical therapy , medical therapy and orthopedic therapies on motor functions among children with cp . there is a large body of research on botulinum toxin , pallus stimulation , therapeutic electrical stimulation , muscle tendon surgery , walking aids and orthosis , hippotherapy , strengthening walking exercises6 . in this study , mean gmfm score was 34.02 28.95 at the time of admission and it increased significantly to 41.08 28.55 at the time of discharge . this result indicates that rehabilitation during hospitalization was effective at improving motor function . furthermore , there was a statistically significant difference between the 1-month and 3-month follows - up compared with the admission total score . there was a significant difference between discharge total score and 1-month and 3-month follow up . this suggests that the information and home - based exercise training provided for caregivers in order to enhance daily living activities might have maintained the significant increase in gmfm total score comparing with admission . decrease in gmfm at 1st month comparing with discharge could be the adaptation of home again . however , the increase in total scores between the 1-month and 3-month follows - up was not significant . it can be inferred from the finding that the mothers were motivated and eager in the first month after discharge from hospital , but subsequently lost motivation . in a previous turkish study , analyzed gmfm scores at the time of admission and discharge among children hospitalized with cp . their study reported mean age , female / male ratio and mean hospitalization stay similar to those of the present study22 . similarly , there was a significant increase in gmfm and discharge total and sub - group scores . these findings suggest that rehabilitation programs have a positive impact on the gross motor functions of children . there is a large body of research on the effects of various treatment methods for cp , many of which have investigated methods to reduce spasticity . total scores increased significantly among children who received 6-weeks of bobath therapy23 . in another study that used gmfm , patients who received a combination of physiotherapy , hypnotherapy analysis of two different groups that received either intensive therapy or routine physiotherapy showed no long - term difference in gmfm . although that study had a different aim , it resembles the present study in terms of evaluation of rehabilitation results and the use of gmfm ; patients received physical therapy as outpatients ; and the use of 18-month follow - up enabled the researchers to observe long - term outcomes . patients were allocated to sub - groups according to calendar age and the denver development test age . patients were evaluated according to the gmfcs with respect to cp type22 . due to the small number of patients in the present study , and since gmfcs provides better indication of motor development , we preferred not to make comparisons according to cp type ; instead , our patients were allocated to monitoring sub - groups according to the gmfcs . the present study aimed to analyze the distribution in groups rather than measuring changes in motor function . the limitations of our study include : 1 ) the children in this study had different types of cp and gmfcs levels , so we were comparing the rehabilitation results of a heterogeneous group . however , all of the participants were children diagnosed with cp who were treated via physiotherapy as inpatients . furthermore , the literature contains other studies that included participants with different gmfcs levels and different types of cp24 . however , an upper limit was set according to bobath , and the duration of hospitalization was determined by a physiatrist supervisor who was unaware of which patients were included in the study . 3 ) the study included no control group ; however , potential delays associated with such a methodology mean that it is ethically inappropriate to exclude a child with cp from rehabilitation for a period longer than 3 months . in light of the research findings , and considering the limitations of similar studies , there is a need for further randomized controlled studies with more activity and participation parameters and a larger sample of cp patients .

[ purpose ] to evaluate the results of bobath - based rehabilitation performed at a pediatric cerebral palsy ( cp ) inpatient clinic . [ subjects and methods ] the study subjects were 28 children with cp who were inpatients at a pediatric service . inclusion criteria were : being an inpatient of our hospital aged 212 with a diagnosis of cp ; having one permanent primary caregiver ; and the caregiver having no medical or psychotic problems . all of the patients received bobath treatment for 1 hour per day , 5 days a week . the locomotor system , neurologic and orthopedic examination , gross motor function measure ( gmfm ) of the patients , and short form-36 ( sf-36 ) of permanent caregivers were evaluated at the time of admission to hospital , discharge from hospital , and at 1 and 3 months after discharge . [ results ] post - admission scores of gmfm at discharge , and 1 and 3 months later showed significant increase . social function and emotional role subscores of sf-36 had increased significantly at discharge . [ conclusion ] bobath treatment is promising and randomized controlled further studies are needed for rehabilitation technics .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC2413076

in the article entitled use of a thin - section archive and enterprise 3d software for long - term storage of thin - slice ct datasets in a previous issue of the journal of digital imaging,1 the authors describe their view of a set - up in which a separate thin - section archive is located at the 3-dimensional server . the crippling effect on current picture archiving and communication system ( pacs ) of the vast increase of data produced by the latest generation computed tomography ( ct ) devices is one of their major concerns . they state that many institutions were forced to devise strategies for handling thin - slice ct data besides their normal data flow . in their solution they also use this concept and only store the thin - slice data on a separate server with limited storage capacity . the phased introduction of thin - slice ct data the authors propose is very interesting and provides a sound solution . at the university medical center groningen , groningen in the netherlands , an approach has been adopted from the start right after the first multidetector ct ( mdct ) was placed including one large pacs containing all data including the thin - slice ct data sets , a fully integrated 3d - server - based rendering system , and an enterprise webserver containing limited and nonpermanent data . this shows that phase 3 as stated by meenan et al.1 could very well be the first step when one is able to employ the right tools . in one step , we went to full usage and storage of all available thin - slice data from our ct systems , which would correspond with phase 3 as proposed by meenan et al.1 this means that every image acquired and reconstructed is actually stored on the pacs for review including all thin slices . currently , a rox5 picture archiving and communication system ( pacs ) ( rogan delft , veenendaal , the netherlands ) is installed at our department . this is an everything on - line ( eol ) pacs with 21 tb of harddisk storage and 20 tb of near - line backup storage on two dvd jukeboxes . remaining dvds are now stored off - line , but transition to ultra density optical ( udo ) disks will be made soon providing a fully available near - line storage again . the eol concept provides all data , including thin slices , on the direct available storage of the pacs . the near - line storage on dvd is purely for backup and security reasons and will not be accessed for retrieval in normal working conditions of the pacs . our 3d server is an aquariusnet server ( tera - recon , san mateo , ca ) with 0.7 tb of storage and two volpro rendering boards and allows the 3d rendering of a maximum of 3,500 ct slices at once . for distribution of patient image data throughout the hospital , an enterprise webserver is installed ( web-1000 , agfa , belgium ) with a short - term storage of 2 tb . all ct modalities are configured such that they automatically reconstruct and store both thin - slice and thick - slice data sets and push them to the pacs . our router ( rogrouter , rogan delft , veenendaal , the netherlands ) will automatically route the incoming data to different locations such as the institutional webserver . only a series of less than 300 slices are stored in the institutional webserver , whereas all data , including thin - slices , are sent to the pacs ( fig . 1 ) . fig 1data acquired by the ct scanner , including thin slices , are sent to the pacs through the router . all data are stored in the pacs and the near - line backup dvd . through the aquariusnet , the institution - wide webserver only receives series below the threshold of 300 images per series . data acquired by the ct scanner , including thin slices , are sent to the pacs through the router . all data are stored in the pacs and the near - line backup dvd . through the aquariusnet , the institution - wide webserver only receives series below the threshold of 300 images per series . our pacs viewer ( hyperview pro , rogan delft , veenendaal , the netherlands ) is a basic viewer , which has only functionality to visualize and manipulate images , either acquired or reconstructed results ( secondary captures ) . the thin - client post processing software used for integration into the radiological work stations is called aquariusnet ( terarecon , san mateo , ca ) . this is a commercially available , server - based solution , which brings interactive 3d rendering and manipulation to the physician s desktop without the requirement of specialized hardware . the rendering is performed on the processing server : all data remain at the server ( no transmission of large data sets throughout the hospital ) and only the rendered and manipulated images are displayed in the client software at the radiological work station ( fig . 2 ) . the central server can be managed and controlled locally through a secure , password protected webpage . after login to the server webpage , full control of the system is possible , even allowing a remote restart of the entire system . the pacs viewer is used to access images , which are retrieved to the work station from the pacs using digital imaging and communication in medicine ( dicom ) query / retrieve . the thin - client postprocessing software thin - client application provides series information and user commands to the thin - client postprocessing software server , which actively queries the pacs and renders the data . only resulting images are transferred to the work station and results can be stored into the pacs as dicom secondary capture . the interaction between the pacs viewer and the thin - client postprocessing software thin - client application is established and information about the series , which should be retrieved , is communicated . the pacs viewer is used to access images , which are retrieved to the work station from the pacs using digital imaging and communication in medicine ( dicom ) query / retrieve . the thin - client postprocessing software thin - client application provides series information and user commands to the thin - client postprocessing software server , which actively queries the pacs and renders the data . only resulting images are transferred to the work station and results can be stored into the pacs as dicom secondary capture . the interaction between the pacs viewer and the thin - client postprocessing software thin - client application is established and information about the series , which should be retrieved , is communicated . the integration of the standard pacs viewer and the thin - client post processing viewer was developed using a simple executable call from within the pacs viewer . with this call , the instanceuid of the current series displayed in the pacs viewer is transmitted to the thin - client postprocessing viewer after which the same series is actively requested by the thin - client post processing viewer from the pacs through the post processing server ( fig . 2 ) . all integrations were established by the vendors of our pacs ( rogandelft , veenendaal , the netherlands ) and the thin - client post processing software ( terarecon , san mateo , ca ) . postprocessing results are not archived as a default , but upon request of the radiologist any image or image sequence can be stored in digital imaging and communication in medicine ( dicom ) secondary capture format into the pacs system ( fig . 2 ) . the system was evaluated by presenting a small questionnaire to our ct and mr radiologists . in this questionnaire the following questions were asked : are you using aquariusnet?if yes , for which modality ( ct / mr ) and how often per day?has the use of aquariusnet increased the use of 3d in daily practice when compared with dedicated work stations in terms of a lower threshold to use 3da higher frequency of 3d usedo you still use dedicated 3d work stations ? if yes , for what reason?which grade would you give on a 110 scale ( in which 1 is very bad and 10 is very good ) for ease of usequality of images / rendering are you using aquariusnet ? if yes , for which modality ( ct / mr ) and how often per day ? has the use of aquariusnet increased the use of 3d in daily practice when compared with dedicated work stations in terms of a lower threshold to use 3da higher frequency of 3d use a lower threshold to use 3d a higher frequency of 3d use do you still use dedicated 3d work stations ? which grade would you give on a 110 scale ( in which 1 is very bad and 10 is very good ) for ease of usequality of images / rendering quality of images / rendering the results of the questionnaire were collected and evaluated using excel ( microsoft corporation , redmond , wa , usa ) . our pacs viewer ( hyperview pro , rogan delft , veenendaal , the netherlands ) is a basic viewer , which has only functionality to visualize and manipulate images , either acquired or reconstructed results ( secondary captures ) . the thin - client post processing software used for integration into the radiological work stations is called aquariusnet ( terarecon , san mateo , ca ) . this is a commercially available , server - based solution , which brings interactive 3d rendering and manipulation to the physician s desktop without the requirement of specialized hardware . the rendering is performed on the processing server : all data remain at the server ( no transmission of large data sets throughout the hospital ) and only the rendered and manipulated images are displayed in the client software at the radiological work station ( fig . 2 ) . the central server can be managed and controlled locally through a secure , password protected webpage . after login to the server webpage , full control of the system is possible , even allowing a remote restart of the entire system . the pacs viewer is used to access images , which are retrieved to the work station from the pacs using digital imaging and communication in medicine ( dicom ) query / retrieve . the thin - client postprocessing software thin - client application provides series information and user commands to the thin - client postprocessing software server , which actively queries the pacs and renders the data . only resulting images are transferred to the work station and results can be stored into the pacs as dicom secondary capture . the interaction between the pacs viewer and the thin - client postprocessing software thin - client application is established and information about the series , which should be retrieved , is communicated . the pacs viewer is used to access images , which are retrieved to the work station from the pacs using digital imaging and communication in medicine ( dicom ) query / retrieve . the thin - client postprocessing software thin - client application provides series information and user commands to the thin - client postprocessing software server , which actively queries the pacs and renders the data . only resulting images are transferred to the work station and results can be stored into the pacs as dicom secondary capture . the interaction between the pacs viewer and the thin - client postprocessing software thin - client application is established and information about the series , which should be retrieved , is communicated . the integration of the standard pacs viewer and the thin - client post processing viewer was developed using a simple executable call from within the pacs viewer . with this call , the instanceuid of the current series displayed in the pacs viewer is transmitted to the thin - client postprocessing viewer after which the same series is actively requested by the thin - client post processing viewer from the pacs through the post processing server ( fig . 2 ) . all integrations were established by the vendors of our pacs ( rogandelft , veenendaal , the netherlands ) and the thin - client post processing software ( terarecon , san mateo , ca ) . postprocessing results are not archived as a default , but upon request of the radiologist any image or image sequence can be stored in digital imaging and communication in medicine ( dicom ) secondary capture format into the pacs system ( fig . the system was evaluated by presenting a small questionnaire to our ct and mr radiologists . in this questionnaire the following questions were asked : are you using aquariusnet?if yes , for which modality ( ct / mr ) and how often per day?has the use of aquariusnet increased the use of 3d in daily practice when compared with dedicated work stations in terms of a lower threshold to use 3da higher frequency of 3d usedo you still use dedicated 3d work stations ? if yes , for what reason?which grade would you give on a 110 scale ( in which 1 is very bad and 10 is very good ) for ease of usequality of images / rendering are you using aquariusnet ? if yes , for which modality ( ct / mr ) and how often per day ? has the use of aquariusnet increased the use of 3d in daily practice when compared with dedicated work stations in terms of a lower threshold to use 3da higher frequency of 3d use a lower threshold to use 3d a higher frequency of 3d use do you still use dedicated 3d work stations ? which grade would you give on a 110 scale ( in which 1 is very bad and 10 is very good ) for ease of usequality of images / rendering quality of images / rendering the results of the questionnaire were collected and evaluated using excel ( microsoft corporation , redmond , wa , usa ) . the thin - client postprocessing software s rendering capabilities are used by our radiologists both for magnetic resonance imaging ( mri ) and ct . after the patient data acquisition is complete , the study is available on the main pacs archive within minutes . the radiologist selects the series , which should be viewed in the thin - client postprocessing software viewer in the pacs viewer window and ( simply ) presses the thin - client postprocessing software button . hereafter , the data are actively requested by the thin - client postprocessing software server from the pacs and transferred . within a very short time ( if available on the thin - client postprocessing software server < 2 sec ; if retrieved from the pacs < 2 min ) the default 3d rendering and multi planar reformation ( mpr ) is displayed for the selected data set . of these , 92% ( n = 12 ) uses the postprocessing capabilities frequently and 8% ( n = 1 ) uses it every now and then . most respondents use it for ct only ( 69% ) , whereas the others use it for both ct and mr ( 31% ) . the mean of the usage results is 12 times per day , in which five of the respondents indicate that they use it for every ct study they read . according to 92% ( n = 12 ) of the respondents , the threshold for using 3d is graded lower and the frequency of use higher compared to using dedicated 3d ( stand alone ) work stations . dedicated 3d work stations are still used frequently by 38% ( n = 5 ) , rarely by 15% ( n = 2 ) , and never by 46% ( n = 6 ) . the main reason for using a dedicated 3d work station is to apply more specialized software packages ( eg , mr mammography evaluation ) or to use more enhanced capabilities ( eg , better and more automated segmentation algorithms ) . both the ease of use and the 3d quality of the thin - client post processing software were graded very high at 8 out of 10 . in the background portion , meenan et al.1 pose a number of challenges of clinical use of thin - slice data sets . first , the storage space required and cost - effective storage are mentioned . in our experience , the storage of all data is easily possible as long as you are capable of purchasing your storage space cost - effectively without high markup of the pacs vendor because of the dedicated use of the storage space . these requirements are high , but by having a dedicated radiology network segment with a 1-gb / s network speed the storage of the data and review within radiology is not a problem . for 3d processing , the use of a thin - client solution effectively decreases the required network bandwidth and the limitation to series with a maximum of 300 slices of the forward to the enterprise web - server further decreases the required bandwidth outside radiology . furthermore , the transition of pacs viewers from thick to thin client solutions using wavelet compression techniques will further decrease the bandwidth requirements . the advantage of using one pacs storage system that includes all available data with a dedicated and fast network eliminates the requirement for workflow strategies . the value of applying 3d rendering techniques to ct and mri data has been described frequently . although discussion has started on the integration of these techniques into the normal workflow , the main drawback of most current implementations remains that the rendering is performed on dedicated , stand alone , 3d work stations . especially with the arrival of digital reporting , rendering must be integrated into the normal radiological workflow . using this set - up , the availability of thin - slice data sets is transparent to the users . because all data are stored in one archive permanently and readily available everywhere , only one work station is required for both normal pacs viewing and advanced 3d viewing , and workflow strategies do not have to be implemented .

current developments in storage solutions , pacs , and client - server systems allow for 3d imaging at the desktop . this can be achieved together with full storage into pacs of all slices , including the very large thin - section ct datasets . this paper describes a possible setup , which has been in operation for several years now , in response to an article by meenan et al . previously published in this journal ( 1 ) .

INTRODUCTION OUR CURRENT SETUP PACS Viewer Post Processing Software Package Usage Evaluation RESULTS DISCUSSION CONCLUSION

PMC3129101

participants for the study were 40 right handed female students ( 21 - 24 years old ) , all reported being free from current or past history of head injuries , mental or neurological disorders and drugs . four affective tune clips ( 3 min ) were selected among cinema and documentary repertoires and presented to subjects in a random order . the average of self reports in a previous study ( likert , scale of 1 - 7 ) have been reported 4.61,4.43 , 4.03 , 5.61 for relaxed , happy , anxious and sad states , respectively.7 study was conducted in an eeg laboratory in the semnan university under supervisory of neurologists during year 2008 . a coupling eye closed awakened eeg was recorded in 16 electrode sites in pre - stimulus ( viewing dark computer screen ) and post - stimulus condition ( viewing movie clips ) . ten 2-s chunks of artifact - free data were analyzed using fast fourier transformation ( fft ) with a hamming window and estimates of absolute spectral power were generated for the alpha band ( 8 - 13hz ) . a guiding assumption underlying the interpretation of findings involving eeg alpha asymmetry participants for the study were 40 right handed female students ( 21 - 24 years old ) , all reported being free from current or past history of head injuries , mental or neurological disorders and drugs . four affective tune clips ( 3 min ) were selected among cinema and documentary repertoires and presented to subjects in a random order . the average of self reports in a previous study ( likert , scale of 1 - 7 ) have been reported 4.61,4.43 , 4.03 , 5.61 for relaxed , happy , anxious and sad states , respectively.7 study was conducted in an eeg laboratory in the semnan university under supervisory of neurologists during year 2008 . a coupling eye closed awakened eeg was recorded in 16 electrode sites in pre - stimulus ( viewing dark computer screen ) and post - stimulus condition ( viewing movie clips ) . ten 2-s chunks of artifact - free data were analyzed using fast fourier transformation ( fft ) with a hamming window and estimates of absolute spectral power were generated for the alpha band ( 8 - 13hz ) . a guiding assumption underlying the interpretation of findings involving eeg alpha asymmetry participants for the study were 40 right handed female students ( 21 - 24 years old ) , all reported being free from current or past history of head injuries , mental or neurological disorders and drugs . four affective tune clips ( 3 min ) were selected among cinema and documentary repertoires and presented to subjects in a random order . the average of self reports in a previous study ( likert , scale of 1 - 7 ) have been reported 4.61,4.43 , 4.03 , 5.61 for relaxed , happy , anxious and sad states , respectively.7 study was conducted in an eeg laboratory in the semnan university under supervisory of neurologists during year 2008 . a coupling eye closed awakened eeg was recorded in 16 electrode sites in pre - stimulus ( viewing dark computer screen ) and post - stimulus condition ( viewing movie clips ) . ten 2-s chunks of artifact - free data were analyzed using fast fourier transformation ( fft ) with a hamming window and estimates of absolute spectral power were generated for the alpha band ( 8 - 13hz ) . a guiding assumption underlying the interpretation of findings involving eeg alpha asymmetry is that greater alpha power is indicative of less cortical activity in broad underling regions.8 significant within subjects effects were revealed for stateside ( wilks lambda = 0.112 , f = 30.651 , p < 0.001 ) and for state effects ( wilks lambda = 0.09 , f = 35.127 , p < 0.001 ) . a significant effect was also found for state in frontal temporal and parietal lobes ( see table 1 ) . consequently , pair wise comparison between affective states , based on estimated marginal means and benferroni adjustment revealed significant differences ( p < 0.001 ) between five states . univariate test for state and stateside effects the baseline - affective states comparison showed significant differences ( p < 0.001 ) for overall frontal ( in happy state ) temporal and parietal lobes ( in happy and sad states ) . results were also revealed significantly right asymmetry ( increase in relative right activity ) ( p < 0.001 ) for temporal ( in all states ) and parietal lobes ( in baseline , happy , anxious and sad states ) . ( see graph 1 ) absolute alpha power means for left and right frontal , temporal and parietal lobes under the baseline and affective states significant within subjects effects were revealed for stateside ( wilks lambda = 0.112 , f = 30.651 , p < 0.001 ) and for state effects ( wilks lambda = 0.09 , f = 35.127 , p < 0.001 ) . a significant effect was also found for state in frontal temporal and parietal lobes ( see table 1 ) . consequently , pair wise comparison between affective states , based on estimated marginal means and benferroni adjustment revealed significant differences ( p < 0.001 ) between five states . univariate test for state and stateside effects the baseline - affective states comparison showed significant differences ( p < 0.001 ) for overall frontal ( in happy state ) temporal and parietal lobes ( in happy and sad states ) . results were also revealed significantly right asymmetry ( increase in relative right activity ) ( p < 0.001 ) for temporal ( in all states ) and parietal lobes ( in baseline , happy , anxious and sad states ) . ( see graph 1 ) absolute alpha power means for left and right frontal , temporal and parietal lobes under the baseline and affective states significant within subjects effects were revealed for stateside ( wilks lambda = 0.112 , f = 30.651 , p < 0.001 ) and for state effects ( wilks lambda = 0.09 , f = 35.127 , p < 0.001 ) . a significant effect was also found for state in frontal temporal and parietal lobes ( see table 1 ) . consequently , pair wise comparison between affective states , based on estimated marginal means and benferroni adjustment revealed significant differences ( p < 0.001 ) between five states . the baseline - affective states comparison showed significant differences ( p < 0.001 ) for overall frontal ( in happy state ) temporal and parietal lobes ( in happy and sad states ) . results were also revealed significantly right asymmetry ( increase in relative right activity ) ( p < 0.001 ) for temporal ( in all states ) and parietal lobes ( in baseline , happy , anxious and sad states ) . ( see graph 1 ) absolute alpha power means for left and right frontal , temporal and parietal lobes under the baseline and affective states bilateral decrease activity of the frontal lobe during the happy state could be interpreted as variability of overall frontal region to happy state and was congruent with those who have emphasized on bilateral variations in brain regions under affective states.34 the parietal and temporal lobes variability in happy and anxious states ( decrease in activity ) were also suggested these as important regions related to positive and negative effect . the right asymmetry in temporal and parietal lobe during most affective states were highlighted right posterior activity as an important function related to affective states as well . these findings were compatible with those who emphasized on the role of right posterior regions in affective states.56 overall variability of frontal , temporal and parietal lobes as well as relative right temporal and parietal activity ( right asymmetry ) seems to mediate positive and negative affective states . shmh has conducted the research , supervised all parts of the experiment and prepared the manuscript . st and ib have coordinated in the statistical analysis and provided important technical support for the experiment .

background : studies have emphasized on the frontal eeg asymmetry as a mediator of emotional states . this research was aimed to examine the overall and asymmetric activities of the brain lobes under the affective states.methods:a coupling eeg was recorded in an eye closed awakened condition from 40 right handed female students under the baseline and affective states.results:bilaterally decreased activities in anterior and posterior regions and also a superimposed right asymmetry in posterior regions were revealed.conclusions:variability of frontal , temporal and parietal lobes together with a superimposed relative right posterior activity may mediate affective states .

Methods None Participants Affective Stimuli Procedure Results None State and State Overall and Asymmetric Activity Conclusions Authors Contributions

PMC4520505

bland - white - garland ( bwg ) syndrome is a congenital heart defect in which the left coronary artery abnormally branches off the pulmonary trunk . its prevalence is approximately 1 : 300,000 births , which corresponds to approximately 0.4% of all congenital heart defects [ 1 , 2 ] . it is the most frequent cause of myocardial infarction in children ; mortality among untreated individuals reaches 90% , while the remaining 10% survive due to the existence of collateral circulation from the right coronary artery . depending on the extent of the connections between the coronary arteries , bwg syndrome may present itself at elderly age or remain asymptomatic throughout the patient 's life [ 2 , 3 ] . symptoms of congestive heart failure , myocardial ischemia , left ventricular contractility impairments , or impairments in the functioning of the mitral valve the syndrome 's subjective manifestations may include dyspnea , fatigability , tearfulness , or paleness [ 2 , 4 ] . the present study presents cases of two children in whom extracorporeal membrane oxygenation ( ecmo ) was employed after the surgical repair of bwg syndrome and one child in whom ecmo was not required after this procedure . a 12-year - old boy diagnosed with bwg syndrome and secondary dilated cardiomyopathy was admitted in order to undergo surgical treatment . physical examination revealed a systolic murmur above the apex of the heart ; no other abnormalities were found . left ventricular contractility was in the lower range of normal levels . retrograde coronary blood flow was observed from the opening of the left coronary artery in the pulmonary trunk . the location of the coronary opening on the posterolateral wall of the pulmonary trunk precluded coronary artery transplantation . therefore , using extracorporeal circulation and takeuchi 's procedure , a tunnel was formed inside the pulmonary artery from the outlet of the left coronary artery to the created aortopulmonary window . a 4-month - old girl diagnosed with a hemodynamically significant defect bwg syndrome , third - degree mitral valve insufficiency , and left ventricular heart failure was admitted in order to undergo a repair procedure . preoperative electrocardiography ( ecg ) revealed st - segment elevation in precordial leads v5 and v6 as well as elevation of cardiac markers . the cardiac ejection fraction ( ef ) 38% , and left ventricular end - diastolic ( lved ) diameter at approx . cardiac catheterization demonstrated that the trunk of the left coronary artery branched off the posterolateral wall of the pulmonary trunk , which , considering the distance , precluded the implantation of a vascular graft into the aorta . the procedure was performed under extracorporeal circulation ; the pulmonary artery was incised transversely above the origin of the left coronary artery ; subsequently , a strip of the pulmonary trunk was excised together with the outlet of the left coronary artery . a strip of the aorta , pedicled on the posterior wall , was incised and sutured to the pulmonary trunk strip , creating a tunnel from the outlet of the left coronary artery to the ascending aorta , running outside the pulmonary trunk . the defect in the wall of the pulmonary trunk was repaired with preserved bovine pericardium . postoperatively , on the day of the procedure , significant left ventricular failure was observed along with impairment of contractility a decision was made to introduce mechanical left ventricular support using venous - arterial extracorporeal circulation . on the 4 postoperative day , circulatory support was disconnected . due to pneumonitis , the patient remained in the intensive care unit until the 24 postoperative day . a control cardiac echo performed at discharge from the icu demonstrated ef of 32% , lved diameter of 36 mm , and mitral insufficiency reduced to the second degree . during the following days , the clinical condition of the child improved as the indices of inflammation subsided systematically . , the patient was transferred to the pediatric cardiology ward , where she remained for the next four weeks . she was discharged home on the 51 postoperative day in good general condition and stable circulatory and respiratory status . although cardiac ultrasonography revealed generalized left ventricular hypokinesia , it also demonstrated a significant improvement of the ejection fraction ( ef = 48% ) in comparison to previous examinations ; the mitral insufficiency was still present , but its gradual regression was observed . a 6-month - old girl with body mass of 4.6 kg was referred to our center in order to undergo surgical repair of bwg syndrome . her records from the previous hospital included the diagnosis of third - degree mitral insufficiency and left ventricular heart failure . the ejection fraction was 18% ; left ventricular dilatation with an akinetic apical aneurysm was observed . as in the case of the previous patient , cardiac catheterization demonstrated that the whole left coronary artery branched off the posterolateral wall of the pulmonary trunk . the performed procedure employed the same technique that was used in the case of the previous patient and also involved the closure of asd . therefore , a decision was made to introduce mechanical circulatory support instead of extracorporeal circulation and to maintain it during the postoperative period . on the 2 postoperative day , cardiac echo demonstrated ef of 19% , lved diameter of 36 mm , and a reduction of mitral insufficiency from the third to second degree . on the 3 postoperative day on the 6 day , neurological symptoms appeared in the form of pathological muscle tension and eye movements to the right side . at this point , the patient underwent computed tomography of the head . the examination revealed the presence of fresh diffuse ischemic lesions in both temporal , frontal , and occipital lobes ( caused by numerous embolisms originating from the akinetic left ventricular apex ) . on the 30 day , the patient was transferred to the pediatric cardiology ward , where she continued to undergo catecholamine therapy . a systematic regression of neurological symptoms was noted , but left - sided hemiparesis persisted . after further seven weeks of treatment , the patient was discharged home in good general condition and stable circulatory and respiratory status . regular follow - up visits to cardiology and neurology clinics were recommended to the patient . the following techniques are reported to be feasible in this context : creating a bypass from the aorta to the left coronary artery or the descending branch of the left coronary artery ( lad ) , transplanting the left coronary artery along with its outlet from the pulmonary trunk to the aorta , or forming a tunnel from the wall of the pulmonary trunk , connecting the left coronary artery with the aorta and running inside the pulmonary trunk using takeuchi 's method or outside the pulmonary trunk . the method which appears to offer best results consists in transplanting the left coronary artery to the aorta . its use is limited by anatomic variants which preclude repairing the defect in this manner [ 5 , 6 ] . in this situation , takeuchi 's technique may be used , or a connecting tunnel may be created outside the pulmonary trunk [ 5 , 6 ] . this method of repair was used in patients 2 and 3 , while takeuchi 's procedure was employed in patient 1 . this technique offers very good early results , but is sometimes followed by long - term complications such as pulmonary artery stenosis or shunt from the tunnel to the lumen of the pulmonary trunk . although patients 2 and 3 exhibited third - degree mitral insufficiency , no repair procedure was attempted , as the insufficiency was caused by generalized hypokinesia and left ventricular dilatation . in such cases , it is difficult to assess the actual degree of insufficiency . although mechanical circulatory support employing extracorporeal circulation has been used for many years in the treatment of heart and lung diseases , clear qualifying criteria for its use are still lacking . the primary reasons for its use in children include a low ejection fraction after cardiac surgery procedures [ 7 , 8 ] . the most significant complications resulting from using this method include bleeding , sepsis , and ischemic or hemorrhagic changes within the central nervous system . longer duration of therapy entails a lower chance of recovery and a higher risk of complications . the subject literature features reports of cases similar to ours in that they required the use of mechanical circulatory support after the surgical repair of bwg syndrome [ 1 , 6 ] . despite the risk of numerous complications , mechanical circulatory support offers a chance of survival to patients with a low left ventricular ejection fraction . as this is a frequent problem after the surgical repair of bwg syndrome , the authors suggest that such repair should only be performed in centers that are able to provide mechanical circulatory support .

the anomalous origin of the left coronary artery arising from the pulmonary artery ( alcapa ) , also known as bland - white - garland ( bwg ) syndrome , is a rare congenital heart disease . we present cases of three children in whom bwg syndrome was repaired surgically . in two of them , the left coronary artery was transplanted from the pulmonary trunk to the aorta , and in one , the takeuchi procedure was performed . in both cases in which the left coronary artery was transplanted to the aorta , mechanical circulatory support was used after the surgery . this was due to a low ejection fraction ( 10% ) while weaning from cardiopulmonary bypass . although associated with numerous complications , mechanical circulatory support can be a lifesaving therapy in patients with a poor left ventricular function after the correction of bwg syndrome .

Introduction Case 1 Case 2 Case 3 Discussion Conclusions Disclosure

PMC3013731

the ability of certain dna sequences to adopt alternative conformations , in addition to the canonical watson indeed , a large number of studies have documented the formation of alternative ( non - b ) dna structures by biophysical methods , including x - ray crystallography ( 24 ) , nuclear magnetic resonance ( nmr ) spectroscopy ( 5 ) and circular dichroism ( 6 ) . other methods , such as the detection of single - stranded bases upon non - b dna structure formation by chemical and enzymatic probes and the relaxation of negative supercoiling by two - dimensional gel electrophoresis have played a major role in revealing the formation of non - b dna conformations in biological systems ( 79 ) . , inverted repeats can adopt cruciform structures , runs of alternating purine pyrimidine bases are able to switch from the right - handed b- to the left - handed z - dna helix , homo(purinepyrimidine ) tracts with mirror repeat symmetry may fold into several types of intramolecular triplexes , four sets of three , four or five guanines , each interrupted by 17 bases , can form highly stable , polymorphic , quadruplex structures and direct repeats can give rise to loops or hairpins through the misalignment of complementary strands , also known as slipped structures ( 10 ) . a number of bioinformatic searches have been conducted with the aim of identifying the biological relevance of putative non - b dna structures in mammalian and other genomes ( 1 ) . these studies support the notion that the secondary structure conformational domain , rather than the underlying sequence symmetry , often contributes to the control of diverse biological functions , including replication , transcription , immune response ( 11 ) , recombination and antigenic variation in human pathogens ( 1,12 ) . concomitant to this notion , a number of studies have provided circumstantial evidence for the involvement of dna secondary structures in inducing genetic instability , both in model systems ( 1315 ) and in association with human genetic disease ( 1620 ) , including genomic regions that do not contain known genes , suggesting that deeper functional annotation across these regions is warranted . therefore , the need has arisen to provide the scientific community with a tool that offers a systematic cataloguing of all predicted sequences currently known to potentially form alternative dna conformations . the non - b db database bridges this gap by providing a resource for searching , mapping and comparing non - b dna - forming motifs among various mammalian species . to date , several reports have detailed methods aimed at enumerating and evaluating predicted non - b dna - forming elements from genomic sequences , including quadbase ( 21 ) , tts ( 22 ) , trf ( 23 ) and others ( documented at http://nonb.abcc.ncifcrf.gov/resources/ ) . these reports use various consensus - based scanning methods for identifying one specific class of predicted non - b dna structure . in some cases , the identified motifs are screened for the presence of other overlapping functional motifs , such as sp1 binding sites and cpg islands ( 24 ) . in other cases , the resulting motifs can be searched by genomic position and scanned for the presence of other nearby non - b dna predicted features [ e.g. triplex sequences near quadruplexes ( 22 ) ] . more recently , analyses that incorporate thermodynamic values into the overall scoring method ( 2527 ) have been reported . together , these resources provide an important , yet partial , view into the complexities of locating and characterizing the many different sequence motifs that have the potential of forming non - b dna structures . our database expands on these functionalities by including all classes of predicted non - b dna - forming sequences and by using the latest genome assemblies of human , mouse and other mammalian species . the non - b dna data are available with current genomic annotation data and polymorphism information . importantly , non - b db provides the capacity to visualize the data in a genomic context that is fully integrated with other genomic features , such as genes and single - nucleotide polymorphisms ( snps ) . the same interface allows for the users to upload their own annotation data , which are displayed alongside the in - house data through the polybrowse and ucsc interfaces . one of the main difficulties in developing and evaluating algorithms that predict the likely candidates for each class of non - b structures is the lack of large collections of experimental data that have validated their formation in vivo . although most non - b dna structures can be formed under in vitro conditions , the identification of such conformations in vivo and the elucidation of parameters that govern their b to non - b equilibria have presented formidable challenges . in addition , these equilibria are influenced by local superhelical density , the presence of nearby dna unwinding element complexes ( dues ) ( 28 ) , the transcriptional status , nucleosome assembly and other tissue / temporally regulated biological processes . in light of these considerations , we have taken the approach of using rather broad and general identification methods based exclusively on sequence features ; thus , although subsequent filtering of the sampled data is straightforward because of the flexibility provided by the database , our current criteria are expected to include a subset of both false positive and negative hits . we have previously reported the construction of a database containing information on mouse indel polymorphisms ( 30 ) . herein , we have extended that system to include motifs with the potential to form non - b dna structures . a number of studies in vitro ( 3134 ) and in vivo ( 29,3538 ) have indicated that the structural transition from b to non - b dna is assisted by unrestrained negative supercoiling . in mammalian cells , the global steady - state levels of negative supercoiling vary depending on chromosomal location ( 39 ) , but are expected to increase transiently by processes , such as transcription , replication and repair , that entail separation of the complementary strands and thus affect nucleosome occupancy ( 29,38,4042 ) . however , because the kinetics of these processes may vary among cell types and various developmental stages , an assessment of the probability that a defined chromosomal sequence might exist in the non - b form is currently not available . indeed , only limited overlap has been reported between the predicted z - dna formation based on in silico thermodynamic predictions and genomic loci bound to the z domain of adar1 , which displays high specificity for z - dna ( 43 ) . thus , a combination of factors , including nucleosome occupancy , negative supercoiling , matrix attachment sites , replication , transcription and repair may underlie b to non - b equilibria in vivo . in the absence of such information , our search algorithms were based solely on sequence relationships derived from in vitro data . the general approach involves running a scanning application for each specific predicted non - b dna class against each chromosome ( table 1 ) , including g - quadruplex motifs , alternating purine pyrimidine sequences , mirror repeats , inverted repeats and direct repeats . although the mirror repeat class as a whole has not been reported to form specific non - b dna structures , it is included in the database as it is used as a first step in the identification of triplex - forming motifs , i.e. the subset of mirror repeats with purine / pyrimidine content . table 1.criteria for predicting non - b dna - forming motifs in non - b dbdna featuresearch criteriasubset of dna feature forming non - b dnasearch criteria for subset of dna featureinverted repeatrepeat : 10100 ntcruciform motifrepeat : 10100 ntspacer : 0100 ntspacer : 03 ntmirror repeatrepeat : 10100 nttriplex motifrepeat : 10100 ror y ntspacer : 0100 ntspacer : 08 ntdirect repeatrepeat : 1050 ntslipped motifrepeat : 1050 ntspacer : 05 ntspacer : 0 ntz - dna repeat5 units of cg / tg or cg / ca repeatswhole setas per the whole setg - quadruplex forming repeatfour identical blocks of ( 37 ) g nt , each block separated by 17 ntwhole setas per the whole seta - phased repeat3 runs of a - tracts with 10-bp phasingwhole setas per the whole setinverted repeat : a pair of dna sequences , each 10100 nt in length and separated by a spacer of 0100 nt , whose sequence composition on the same strand of dna is such that the bases of the first repeat , when read in the 53 orientation , are complementary to those of the second repeat read in the 35 orientation . the term crick hydrogen bonding scheme , whereby a only pairs with t and c only pairs with g. only perfect inverted repeats that conform to this watson crick pairing scheme are considered.cruciform motif : the subset of inverted repeat sequences in which the spacer comprises 03 bases ; due to their proximity , this subset of inverted repeat sequences may fold - back and form intramolecular , antiparallel , double helices stabilized by watson crick hydrogen bonds , i.e. a cruciform structure ( 1,34).mirror repeat : a pair of dna sequences , each 10100 nt in length and separated by a spacer of 0100 nt , whose sequence composition on the same strand of dna is such that the bases of the first repeat , when read in the 53 orientation , are identical to those of the second repeat read in the 35 orientation ( palindrome ) ; only perfectly matching repeats are included.triplex motif : the subset of mirror repeat sequences comprising only purines ( r = a and g ) [ or pyrimidines ( y = c and t ) ] on the same strand of dna , and which are separated by few ( 08 ) nt ( spacer ) . these motifs are able to form various intramolecular three - stranded ( triplex , h - dna ) isoforms stabilized by hoogsteen hydrogen bonds ( 1,52,53 ) . only ry - containing mirror repeats that may yield a : at and g : gc base triplets ( colon indicates hoogsteen hydrogen bonded bases ; dot indicates watson crick hydrogen bonded bases ) for the r : ry type of intramolecular triplexes and t : at and c : gc triplets for the y : ry type of intramolecular triplexes are included since these are considered the most stable triplet combinations.direct repeat : two tracts of dna , each comprising 1050 nt and separated by 05 nt , having the same sequence composition.slipped motif : the subset of direct repeat sequences without a spacer ( tandem repeats ) ; when aligned in an out - of - register fashion , tandem repeats may give rise to single - stranded loops and/or hairpins ( 1).z - dna motif : five or more tandem repeats , each comprising an alternating pyrimidine purine dinucleotide motif , in which the pattern yg is maintained on at least one of the dna strands ; examples include ( cgcg)6 , ( catg)5 and [ ( tg)3(cg)4(cg)4(ca)3 ] ; these motifs may adopt the left - handed z - dna conformation ( 3,54).g - quadruplex - forming repeat : four blocks , each containing the same number ( n ) of g bases ( n can vary from 3 to 7 ) , on the plus or minus strand , separated by 17 nt ; this type of dna sequence may adopt quadruplex structures ( 2 ) ; overlapping tracts of four g - blocks are also considered.a - phased repeat : three runs of a bases ( a - tracts ) in phase with the helical pitch of the dna double - helix , i.e. 10 bp ; an a - tract is defined as a set of at base - pairs without a tpa step ( 47,5557 ) ; three or more tracts of a37 , t37 , aaattt , aaatttt and aaaattt ( in any combination ) on the plus or minus strand , whose centers are separated by 10 bases , are considered ; since a - tracts induce static bends in the dna double helix , the overall dna superhelix is expected to display either a left - handed or a right - handed writhe ( 47,5557 ) ; as mentioned , all the search criteria used herein do not allow for interruptions in the repeats and no thermodynamic information was factored - in in the algorithms used . criteria for predicting non - b dna - forming motifs in non - b db inverted repeat : a pair of dna sequences , each 10100 nt in length and separated by a spacer of 0100 nt , whose sequence composition on the same strand of dna is such that the bases of the first repeat , when read in the 53 orientation , are complementary to those of the second repeat read in the 35 orientation . the term crick hydrogen bonding scheme , whereby a only pairs with t and c only pairs with g. only perfect inverted repeats that conform to this watson spacer comprises 03 bases ; due to their proximity , this subset of inverted repeat sequences may fold - back and form intramolecular , antiparallel , double helices stabilized by watson crick hydrogen bonds , i.e. a cruciform structure ( 1,34 ) . mirror repeat : a pair of dna sequences , each 10100 nt in length and separated by a spacer of 0100 nt , whose sequence composition on the same strand of dna is such that the bases of the first repeat , when read in the 53 orientation , are identical to those of the second repeat read in the 35 orientation ( palindrome ) ; only perfectly matching repeats are included . triplex motif : the subset of mirror repeat sequences comprising only purines ( r = a and g ) [ or pyrimidines ( y = c and t ) ] on the same strand of dna , and which are separated by few ( 08 ) nt ( spacer ) . these motifs are able to form various intramolecular three - stranded ( triplex , h - dna ) isoforms stabilized by hoogsteen hydrogen bonds ( 1,52,53 ) . only ry - containing mirror repeats that may yield a : at and g : gc base triplets ( colon indicates hoogsteen hydrogen bonded bases ; dot indicates watson crick hydrogen bonded bases ) for the r : ry type of intramolecular triplexes and t : at and c : gc triplets for the y : ry type of intramolecular triplexes are included since these are considered the most stable triplet combinations . direct repeat : two tracts of dna , each comprising 1050 nt and separated by 05 nt , having the same sequence composition . slipped motif : the subset of direct repeat sequences without a spacer ( tandem repeats ) ; when aligned in an out - of - register fashion , tandem repeats may give rise to single - stranded loops and/or hairpins ( 1 ) . z - dna motif : five or more tandem repeats , each comprising an alternating pyrimidine purine dinucleotide motif , in which the pattern yg is maintained on at least one of the dna strands ; examples include ( cgcg)6 , ( catg)5 and [ ( tg)3(cg)4(cg)4(ca)3 ] ; these motifs may adopt the left - handed z - dna conformation ( 3,54 ) . g - quadruplex - forming repeat : four blocks , each containing the same number ( n ) of g bases ( n can vary from 3 to 7 ) , on the plus or minus strand , separated by 17 nt ; this type of dna sequence may adopt quadruplex structures ( 2 ) ; overlapping tracts of four g - blocks are also considered . a - phased repeat : three runs of a bases ( a - tracts ) in phase with the helical pitch of the dna double - helix , i.e. 10 bp ; an a - tract is defined as a set of at base - pairs without a tpa step ( 47,5557 ) ; three or more tracts of a37 , t37 , aaattt , aaatttt and aaaattt ( in any combination ) on the plus or minus strand , whose centers are separated by 10 bases , are considered ; since a - tracts induce static bends in the dna double helix , the overall dna superhelix is expected to display either a left - handed or a right - handed writhe ( 47,5557 ) ; as mentioned , all the search criteria used herein do not allow for interruptions in the repeats and no thermodynamic information was factored - in in the algorithms used . the output file in gff format ( http://nonb.abcc.ncifcrf.gov/faqs/ ) the data from all such scans are merged and can be queried and displayed using our local instance of gbrowse called polybrowse ( 44 ) at http://pbrowse3.abcc.ncifcrf.gov/cgi-bin/gb2/gbrowse/human_37 and several gff - based query tools at http://nonb.abcc.ncifcrf.gov ( figure 1 ) . importantly , the result pages produced from the queries contain links that allow the user to switch to the genome browser view of that feature , as well as a view that provides the sequence and other annotations for each feature . non - b db user interface ( a ) three query modes are available at the genomic database search tools page of the non - b db web interface : search by feature ( shown ) , search by feature attributes , and search by location ( a feature browser ) . in this example , all non - b dna motifs were queried by the gene symbol myc . ( b ) query results and links to polybrowse , ucsc genome browser , biodbnet ( 51 ) , etc . users may download the results in gff format and tab delimited format . direct access to the sequence and other annotation information for each of the features is available by clicking on the non - b db user interface ( a ) three query modes are available at the genomic database search tools page of the non - b db web interface : search by feature ( shown ) , search by feature attributes , and search by location ( a feature browser ) . in this example , all non - b dna motifs were queried by the gene symbol myc . ( b ) query results and links to polybrowse , ucsc genome browser , biodbnet ( 51 ) , etc . direct access to the sequence and other annotation information for each of the features is available by clicking on the a link ( red box ) . these data represent the basis for the non - b dna annotation information for each species . the scanning criteria do not allow for mismatches within the repeat segments ; however , this feature may be added as information becomes available as to the acceptable structural tolerances for each mismatch case . also , currently not included are very large palindromes ( > 100 kb ) , such as those that characterize the y chromosome and whose recombination is known to lead to spermatogenic failure ( 45,46 ) . nevertheless , some aspects related to the presence of mismatches are presented in the polymorphism analysis described below . after scanning across different mammalian genomes , the numbers of each of the predicted classes of non - b dna structure - forming motifs appear to be quite variable ( table 2 ) . table 2.statistics for dna repeats and non - b dna - forming motifs in non - b dbdna featurehuman 37mouse 37dog 2chimp 2macaque 1g - quadruplex forming repeat374 545559 280492 535314 171298 142inverted repeat1 044 533801 242814 080998 249843 889cruciform motif197 910188 532172 032190 736128 334direct repeat871 0451 593 107968 955787 335765 798slipped motif347 969695 150404 750314 516305 285mirror repeat16 51 7233 431 4861 829 86714 85 13514 55 025triplex motif1 79 623618 928336 6421 05 6401 40 580z - dna repeat294 320690 276261 012278 928280 982a - phased repeat1 130 7319 09 6531 241 0821 085 5911 098 030for the current releases of the five mammalian genomes indicated , the motif searches were performed and the number of features for each class was counted . according to table 1 , the cruciform motifs represent a subset of the inverted repeat class , the slipped motifs represent a subset of the direct repeat class and the triplex motif represents a subset of the mirror repeat class . statistics for dna repeats and non - b dna - forming motifs in non - b db for the current releases of the five mammalian genomes indicated , the motif searches were performed and the number of features for each class was counted . according to table 1 , the cruciform motifs represent a subset of the inverted repeat class , the slipped motifs represent a subset of the direct repeat class and the triplex motif represents a subset of the mirror repeat class . as the overall base composition between different mammalian genomes is rather similar ( data not shown ) , the observed differences in the numbers of predicted non - b dna motifs could simply result from the altered arrangement of bases from one species to another . alternatively , variations in the population of classes of repetitive elements ( sine , line , etc . ) among species , or other unknown features , might also contribute to the observed differences . this interspecies variability appears to be uniformly distributed along the entire chromosomes , rather than concentrated in large repetitive clusters ( data not shown ) . whether these differences play any role or contribute to conferring species - specific differences remains to be investigated . a caveat concerning the simple assessment and comparison of the number of non - b dna - forming repeats among species relates to the criteria used and the counting method . for example , in the g - quadruplex forming sequences , the pattern of a run of 3gs followed by 17 bases repeated four times can be extended , as long as more runs of gs are encountered , resulting in a single cluster that has the potential to form many substructures . although our approach identifies this finding as a single cluster in the database , separate database tables are provided , in which all possible permutations of the sequence that satisfies the consensus sequence are reported . in addition to the non - b dna predicted motifs , the database contains other features of the dna , such as phased a - tracts that impart static bends to the double - helix and may be involved in nucleosome assembly ( 47 ) , simple tandem repeats ( str ) including triplet repeats whose expansions cause a number of neuromuscular disorders ( 20 ) and poly(purinepyrimidine ) tracts , which are characterized by high stacking interactions ( 48 ) . in addition , ncbi - derived features , such as genes , snps and repeatmasker ( http://www.repeatmasker.org/ ) elements are also included . this integrated information is critical not only for guiding the user visually , but also for enabling queries that combine classes , such as exons containing predicted z - dna forming sequences , etc . one of the main features of the non - b db is the ability to compare different mammalian genomes for the presence of non - b dna - forming motifs . figure 2 illustrates this capability by comparing the presence of g - quadruplex forming motifs in the region upstream of the myc locus across the human , chimp , macaque , dog and mouse reference genomes . in order to view syntenic regions in other genomes , the liftover application from the ucsc website areas where a syntenic match failed to be identified ( i.e. that region was absent in the other genome , or mapped redundantly ) do not show a link to that species . other non - b dna tracks available in polybrowse will be described in more detail elsewhere ( cer et . figure 2.(a ) polybrowse view of g - quadruplex forming repeats in the myc gene with species syntenic information . visualization of cross - species information in polybrowse : the genomic region shown in figure 1 is displayed herein to illustrate the syntenic capability . in this case , the g - quadruplex forming repeat track shows the locations of these features in the region near the beginning of the myc gene . the additional track illustrating the liftover1k blocks is also reported . in that track , when the user moves the cursor over the objects , a popup window appears that contains links ( a ) to the syntenic locations in the other available species . ( b e ) results upon clicking on the links for chimp , macaque , mouse and dog , respectively . currently , the liftover feature only allows moving from the human to the other species but not among the non - human species . ( a ) polybrowse view of g - quadruplex forming repeats in the myc gene with species syntenic information . visualization of cross - species information in polybrowse : the genomic region shown in figure 1 is displayed herein to illustrate the syntenic capability . in this case , the g - quadruplex forming repeat track shows the locations of these features in the region near the beginning of the myc gene . the additional track illustrating the liftover1k blocks is also reported . in that track , when the user moves the cursor over the objects , a popup window appears that contains links ( a ) to the syntenic locations in the other available species . ( b e ) results upon clicking on the links for chimp , macaque , mouse and dog , respectively . currently , the liftover feature only allows moving from the human to the other species but not among the non - human species . the computed non - b dna forming elements are likely to be under - represented in our reference genome as their underlying repeats may be polymorphic among individuals . because this type of information may be critical in the context of gene regulation or predisposition to disease ( 48 ) , we used a specific parser to scan both the reference human genome as well as additional sequence sources , such as trace reads from the trace archive ( http://www.ncbi.nlm.nih.gov/traces/trace.cgi ) and contigs ( http://www.ncbi.nlm.nih.gov/projects/wgs/wgsprojectlist.cgi ) from personal genome projects ( 49 ) , for matches to the non - b dna motifs . each match found in either the reference or alternate source is then scored for being polymorphic or not . of the sites identified as polymorphic , a second evaluation is made to determine whether the polymorphism would affect the motif underlying the putative non - b dna structure . the results of this scan are incorporated into the database as a series of separate tracks ( figure 3b , trace gplex tracks ) . additional information can be gathered by extending this type of analysis to sequence alignments using closely related species . currently , only the g - quadruplex forming motif supports this type of query . figure 3.(a ) polybrowse view of some of the non - b dna features in the human myc gene . links to polybrowse and ucsc browsers : ( a ) display of polybrowse rendering the myc gene and its promoter with some of the non - b dna motifs tracks with the pupy [ poly(purinepyrimidine ) ] and polymorphism tracks turned on . several other tracks include features computed at the advanced biomedical computing center ( abcc ) , such as strs , base composition , physical dna characteristics , mapping , as well as the ncbi derived features , including genes , snps , cytogenic markers assembly information , repeatmasker elements , etc . ( b ) display of the myc gene in polybrowse showing some of the polymorphism information in non - b db . below the gene and mrna tracks , the searched motifs in the reference sequence are displayed ( teal ) . an additional track shows specific g - quadruplex motifs in which all of the observed trace files contained mutations disrupting the g - quadruplex motif . ( c ) display of the myc gene at the ucsc human genome browser ( grch37/hg19 assembly ) as linked to non - b db from the search shown in figure 2b . some of the non - b dna motifs from non - b db can be seen in the red rectangular box . ( a ) polybrowse view of some of the non - b dna features in the human myc gene . links to polybrowse and ucsc browsers : ( a ) display of polybrowse rendering the myc gene and its promoter with some of the non - b dna motifs tracks with the pupy [ poly(purinepyrimidine ) ] and polymorphism tracks turned on . several other tracks include features computed at the advanced biomedical computing center ( abcc ) , such as strs , base composition , physical dna characteristics , mapping , as well as the ncbi derived features , including genes , snps , cytogenic markers assembly information , repeatmasker elements , etc . ( b ) display of the myc gene in polybrowse showing some of the polymorphism information in non - b db . below the gene and mrna tracks , the searched motifs in the reference sequence are displayed ( teal ) . an additional track shows specific g - quadruplex motifs in which all of the observed trace files contained mutations disrupting the g - quadruplex motif . ( c ) display of the myc gene at the ucsc human genome browser ( grch37/hg19 assembly ) as linked to non - b db from the search shown in figure 2b . some of the non - b dna motifs from non - b db can be seen in the red rectangular box . in order to provide access to the back - end database the first is a bioperl ( 50 ) set of methods , which is used to query genome databases in various ways , such as by position , by class , or by attribute . this same set of utilities is used within the context of polybrowse ( 44 ) , so that visualization of the genomic features is made available . in addition to linking the outputs from the query tools to the browser for visualization , we also provide links allowing the returned data to be displayed in the familiar ucsc ( http://genome.ucsc.edu/ ) genome browser ( figure 3c ) , as well as links to our biodbnet database warehouse ( 51 ) , which contains gene - centric information derived from several sources , and additional links . to date , several reports have detailed methods aimed at enumerating and evaluating predicted non - b dna - forming elements from genomic sequences , including quadbase ( 21 ) , tts ( 22 ) , trf ( 23 ) and others ( documented at http://nonb.abcc.ncifcrf.gov/resources/ ) . these reports use various consensus - based scanning methods for identifying one specific class of predicted non - b dna structure . in some cases , the identified motifs are screened for the presence of other overlapping functional motifs , such as sp1 binding sites and cpg islands ( 24 ) . in other cases , the resulting motifs can be searched by genomic position and scanned for the presence of other nearby non - b dna predicted features [ e.g. triplex sequences near quadruplexes ( 22 ) ] . more recently , analyses that incorporate thermodynamic values into the overall scoring method ( 2527 ) have been reported . together , these resources provide an important , yet partial , view into the complexities of locating and characterizing the many different sequence motifs that have the potential of forming non - b dna structures . our database expands on these functionalities by including all classes of predicted non - b dna - forming sequences and by using the latest genome assemblies of human , mouse and other mammalian species . the non - b dna data are available with current genomic annotation data and polymorphism information . importantly , non - b db provides the capacity to visualize the data in a genomic context that is fully integrated with other genomic features , such as genes and single - nucleotide polymorphisms ( snps ) . the same interface allows for the users to upload their own annotation data , which are displayed alongside the in - house data through the polybrowse and ucsc interfaces . one of the main difficulties in developing and evaluating algorithms that predict the likely candidates for each class of non - b structures is the lack of large collections of experimental data that have validated their formation in vivo . although most non - b dna structures can be formed under in vitro conditions , the identification of such conformations in vivo and the elucidation of parameters that govern their b to non - b equilibria have presented formidable challenges . in addition , these equilibria are influenced by local superhelical density , the presence of nearby dna unwinding element complexes ( dues ) ( 28 ) , the transcriptional status , nucleosome assembly and other tissue / temporally regulated biological processes . in light of these considerations , we have taken the approach of using rather broad and general identification methods based exclusively on sequence features ; thus , although subsequent filtering of the sampled data is straightforward because of the flexibility provided by the database , our current criteria are expected to include a subset of both false positive and negative hits . we have previously reported the construction of a database containing information on mouse indel polymorphisms ( 30 ) . herein , we have extended that system to include motifs with the potential to form non - b dna structures . a number of studies in vitro ( 3134 ) and in vivo ( 29,3538 ) have indicated that the structural transition from b to non - b dna is assisted by unrestrained negative supercoiling . in mammalian cells , the global steady - state levels of negative supercoiling vary depending on chromosomal location ( 39 ) , but are expected to increase transiently by processes , such as transcription , replication and repair , that entail separation of the complementary strands and thus affect nucleosome occupancy ( 29,38,4042 ) . however , because the kinetics of these processes may vary among cell types and various developmental stages , an assessment of the probability that a defined chromosomal sequence might exist in the non - b form is currently not available . indeed , only limited overlap has been reported between the predicted z - dna formation based on in silico thermodynamic predictions and genomic loci bound to the z domain of adar1 , which displays high specificity for z - dna ( 43 ) . thus , a combination of factors , including nucleosome occupancy , negative supercoiling , matrix attachment sites , replication , transcription and repair may underlie b to non - b equilibria in vivo . in the absence of such information , the general approach involves running a scanning application for each specific predicted non - b dna class against each chromosome ( table 1 ) , including g - quadruplex motifs , alternating purine pyrimidine sequences , mirror repeats , inverted repeats and direct repeats . although the mirror repeat class as a whole has not been reported to form specific non - b dna structures , it is included in the database as it is used as a first step in the identification of triplex - forming motifs , i.e. the subset of mirror repeats with purine / pyrimidine content . table 1.criteria for predicting non - b dna - forming motifs in non - b dbdna featuresearch criteriasubset of dna feature forming non - b dnasearch criteria for subset of dna featureinverted repeatrepeat : 10100 ntcruciform motifrepeat : 10100 ntspacer : 0100 ntspacer : 03 ntmirror repeatrepeat : 10100 nttriplex motifrepeat : 10100 ror y ntspacer : 0100 ntspacer : 08 ntdirect repeatrepeat : 1050 ntslipped motifrepeat : 1050 ntspacer : 05 ntspacer : 0 ntz - dna repeat5 units of cg / tg or cg / ca repeatswhole setas per the whole setg - quadruplex forming repeatfour identical blocks of ( 37 ) g nt , each block separated by 17 ntwhole setas per the whole seta - phased repeat3 runs of a - tracts with 10-bp phasingwhole setas per the whole setinverted repeat : a pair of dna sequences , each 10100 nt in length and separated by a spacer of 0100 nt , whose sequence composition on the same strand of dna is such that the bases of the first repeat , when read in the 53 orientation , are complementary to those of the second repeat read in the 35 orientation . the term complementary refers to the watson crick hydrogen bonding scheme , whereby a only pairs with t and c only pairs with g. only perfect inverted repeats that conform to this watson crick pairing scheme are considered.cruciform motif : the subset of inverted repeat sequences in which the spacer comprises 03 bases ; due to their proximity , this subset of inverted repeat sequences may fold - back and form intramolecular , antiparallel , double helices stabilized by watson crick hydrogen bonds , i.e. a cruciform structure ( 1,34).mirror repeat : a pair of dna sequences , each 10100 nt in length and separated by a spacer of 0100 nt , whose sequence composition on the same strand of dna is such that the bases of the first repeat , when read in the 53 orientation , are identical to those of the second repeat read in the 35 orientation ( palindrome ) ; only perfectly matching repeats are included.triplex motif : the subset of mirror repeat sequences comprising only purines ( r = a and g ) [ or pyrimidines ( y = c and t ) ] on the same strand of dna , and which are separated by few ( 08 ) nt ( spacer ) . these motifs are able to form various intramolecular three - stranded ( triplex , h - dna ) isoforms stabilized by hoogsteen hydrogen bonds ( 1,52,53 ) . only ry - containing mirror repeats that may yield a : at and g : gc base triplets ( colon indicates hoogsteen hydrogen bonded bases ; dot indicates watson crick hydrogen bonded bases ) for the r : ry type of intramolecular triplexes and t : at and c : gc triplets for the y : ry type of intramolecular triplexes are included since these are considered the most stable triplet combinations.direct repeat : two tracts of dna , each comprising 1050 nt and separated by 05 nt , having the same sequence composition.slipped motif : the subset of direct repeat sequences without a spacer ( tandem repeats ) ; when aligned in an out - of - register fashion , tandem repeats may give rise to single - stranded loops and/or hairpins ( 1).z - dna motif : five or more tandem repeats , each comprising an alternating pyrimidine purine dinucleotide motif , in which the pattern yg is maintained on at least one of the dna strands ; examples include ( cgcg)6 , ( catg)5 and [ ( tg)3(cg)4(cg)4(ca)3 ] ; these motifs may adopt the left - handed z - dna conformation ( 3,54).g - quadruplex - forming repeat : four blocks , each containing the same number ( n ) of g bases ( n can vary from 3 to 7 ) , on the plus or minus strand , separated by 17 nt ; this type of dna sequence may adopt quadruplex structures ( 2 ) ; overlapping tracts of four g - blocks are also considered.a - phased repeat : three runs of a bases ( a - tracts ) in phase with the helical pitch of the dna double - helix , i.e. 10 bp ; an a - tract is defined as a set of at base - pairs without a tpa step ( 47,5557 ) ; three or more tracts of a37 , t37 , aaattt , aaatttt and aaaattt ( in any combination ) on the plus or minus strand , whose centers are separated by 10 bases , are considered ; since a - tracts induce static bends in the dna double helix , the overall dna superhelix is expected to display either a left - handed or a right - handed writhe ( 47,5557 ) ; as mentioned , all the search criteria used herein do not allow for interruptions in the repeats and no thermodynamic information was factored - in in the algorithms used . criteria for predicting non - b dna - forming motifs in non - b db inverted repeat : a pair of dna sequences , each 10100 nt in length and separated by a spacer of 0100 nt , whose sequence composition on the same strand of dna is such that the bases of the first repeat , when read in the 53 orientation , are complementary to those of the second repeat read in the 35 orientation . crick hydrogen bonding scheme , whereby a only pairs with t and c only pairs with g. only perfect inverted repeats that conform to this watson cruciform motif : the subset of inverted repeat sequences in which the spacer comprises 03 bases ; due to their proximity , this subset of inverted repeat sequences may fold - back and form intramolecular , antiparallel , double helices stabilized by watson crick hydrogen bonds , i.e. a cruciform structure ( 1,34 ) . mirror repeat : a pair of dna sequences , each 10100 nt in length and separated by a spacer of 0100 nt , whose sequence composition on the same strand of dna is such that the bases of the first repeat , when read in the 53 orientation , are identical to those of the second repeat read in the 35 orientation ( palindrome ) ; only perfectly matching repeats are included . triplex motif : the subset of mirror repeat sequences comprising only purines ( r = a and g ) [ or pyrimidines ( y = c and t ) ] on the same strand of dna , and which are separated by few ( 08 ) nt ( spacer ) . these motifs are able to form various intramolecular three - stranded ( triplex , h - dna ) isoforms stabilized by hoogsteen hydrogen bonds ( 1,52,53 ) . only ry - containing mirror repeats that may yield a : at and g : gc base triplets ( colon indicates hoogsteen hydrogen bonded bases ; dot indicates watson crick hydrogen bonded bases ) for the r : ry type of intramolecular triplexes and t : at and c : gc triplets for the y : ry type of intramolecular triplexes are included since these are considered the most stable triplet combinations . direct repeat : two tracts of dna , each comprising 1050 nt and separated by 05 nt , having the same sequence composition . slipped motif : the subset of direct repeat sequences without a spacer ( tandem repeats ) ; when aligned in an out - of - register fashion , tandem repeats may give rise to single - stranded loops and/or hairpins ( 1 ) . z - dna motif : five or more tandem repeats , each comprising an alternating pyrimidine purine dinucleotide motif , in which the pattern yg is maintained on at least one of the dna strands ; examples include ( cgcg)6 , ( catg)5 and [ ( tg)3(cg)4(cg)4(ca)3 ] ; these motifs may adopt the left - handed z - dna conformation ( 3,54 ) . g - quadruplex - forming repeat : four blocks , each containing the same number ( n ) of g bases ( n can vary from 3 to 7 ) , on the plus or minus strand , separated by 17 nt ; this type of dna sequence may adopt quadruplex structures ( 2 ) ; overlapping tracts of four g - blocks are also considered . a - phased repeat : three runs of a bases ( a - tracts ) in phase with the helical pitch of the dna double - helix , i.e. 10 bp ; an a - tract is defined as a set of at base - pairs without a tpa step ( 47,5557 ) ; three or more tracts of a37 , t37 , aaattt , aaatttt and aaaattt ( in any combination ) on the plus or minus strand , whose centers are separated by 10 bases , are considered ; since a - tracts induce static bends in the dna double helix , the overall dna superhelix is expected to display either a left - handed or a right - handed writhe ( 47,5557 ) ; as mentioned , all the search criteria used herein do not allow for interruptions in the repeats and no thermodynamic information was factored - in in the algorithms used . the output file in gff format ( http://nonb.abcc.ncifcrf.gov/faqs/ ) the data from all such scans are merged and can be queried and displayed using our local instance of gbrowse called polybrowse ( 44 ) at http://pbrowse3.abcc.ncifcrf.gov/cgi-bin/gb2/gbrowse/human_37 and several gff - based query tools at http://nonb.abcc.ncifcrf.gov ( figure 1 ) . importantly , the result pages produced from the queries contain links that allow the user to switch to the genome browser view of that feature , as well as a view that provides the sequence and other annotations for each feature . non - b db user interface ( a ) three query modes are available at the genomic database search tools page of the non - b db web interface : search by feature ( shown ) , search by feature attributes , and search by location ( a feature browser ) . in this example , all non - b dna motifs were queried by the gene symbol myc . ( b ) query results and links to polybrowse , ucsc genome browser , biodbnet ( 51 ) , etc . users may download the results in gff format and tab delimited format . direct access to the sequence and other annotation information for each of the features is available by clicking on the non - b db user interface ( a ) three query modes are available at the genomic database search tools page of the non - b db web interface : search by feature ( shown ) , search by feature attributes , and search by location ( a feature browser ) . in this example , all non - b dna motifs were queried by the gene symbol myc . ( b ) query results and links to polybrowse , ucsc genome browser , biodbnet ( 51 ) , etc . users may download the results in gff format and tab delimited format . direct access to the sequence and other annotation information for each of the features is available by clicking on the these data represent the basis for the non - b dna annotation information for each species . the scanning criteria do not allow for mismatches within the repeat segments ; however , this feature may be added as information becomes available as to the acceptable structural tolerances for each mismatch case . also , currently not included are very large palindromes ( > 100 kb ) , such as those that characterize the y chromosome and whose recombination is known to lead to spermatogenic failure ( 45,46 ) . nevertheless , some aspects related to the presence of mismatches are presented in the polymorphism analysis described below . after scanning across different mammalian genomes , the numbers of each of the predicted classes of non - b dna structure - forming motifs appear to be quite variable ( table 2 ) . table 2.statistics for dna repeats and non - b dna - forming motifs in non - b dbdna featurehuman 37mouse 37dog 2chimp 2macaque 1g - quadruplex forming repeat374 545559 280492 535314 171298 142inverted repeat1 044 533801 242814 080998 249843 889cruciform motif197 910188 532172 032190 736128 334direct repeat871 0451 593 107968 955787 335765 798slipped motif347 969695 150404 750314 516305 285mirror repeat16 51 7233 431 4861 829 86714 85 13514 55 025triplex motif1 79 623618 928336 6421 05 6401 40 580z - dna repeat294 320690 276261 012278 928280 982a - phased repeat1 130 7319 09 6531 241 0821 085 5911 098 030for the current releases of the five mammalian genomes indicated , the motif searches were performed and the number of features for each class was counted . according to table 1 , the cruciform motifs represent a subset of the inverted repeat class , the slipped motifs represent a subset of the direct repeat class and the triplex motif represents a subset of the mirror repeat class . statistics for dna repeats and non - b dna - forming motifs in non - b db for the current releases of the five mammalian genomes indicated , the motif searches were performed and the number of features for each class was counted . according to table 1 , the cruciform motifs represent a subset of the inverted repeat class , the slipped motifs represent a subset of the direct repeat class and the triplex motif represents a subset of the mirror repeat class . as the overall base composition between different mammalian genomes is rather similar ( data not shown ) , the observed differences in the numbers of predicted non - b dna motifs could simply result from the altered arrangement of bases from one species to another . alternatively , variations in the population of classes of repetitive elements ( sine , line , etc . ) among species , or other unknown features , might also contribute to the observed differences . this interspecies variability appears to be uniformly distributed along the entire chromosomes , rather than concentrated in large repetitive clusters ( data not shown ) . whether these differences play any role or contribute to conferring species - specific differences remains to be investigated . a caveat concerning the simple assessment and comparison of the number of non - b dna - forming repeats among species relates to the criteria used and the counting method . for example , in the g - quadruplex forming sequences , the pattern of a run of 3gs followed by 17 bases repeated four times can be extended , as long as more runs of gs are encountered , resulting in a single cluster that has the potential to form many substructures . although our approach identifies this finding as a single cluster in the database , separate database tables are provided , in which all possible permutations of the sequence that satisfies the consensus sequence are reported . in addition to the non - b dna predicted motifs , the database contains other features of the dna , such as phased a - tracts that impart static bends to the double - helix and may be involved in nucleosome assembly ( 47 ) , simple tandem repeats ( str ) including triplet repeats whose expansions cause a number of neuromuscular disorders ( 20 ) and poly(purinepyrimidine ) tracts , which are characterized by high stacking interactions ( 48 ) . in addition , ncbi - derived features , such as genes , snps and repeatmasker ( http://www.repeatmasker.org/ ) elements are also included . this integrated information is critical not only for guiding the user visually , but also for enabling queries that combine classes , such as exons containing predicted z - dna forming sequences , etc . one of the main features of the non - b db is the ability to compare different mammalian genomes for the presence of non - b dna - forming motifs . figure 2 illustrates this capability by comparing the presence of g - quadruplex forming motifs in the region upstream of the myc locus across the human , chimp , macaque , dog and mouse reference genomes . in order to view syntenic regions in other genomes , the liftover application from the ucsc website areas where a syntenic match failed to be identified ( i.e. that region was absent in the other genome , or mapped redundantly ) do not show a link to that species . other non - b dna tracks available in polybrowse will be described in more detail elsewhere ( cer et . figure 2.(a ) polybrowse view of g - quadruplex forming repeats in the myc gene with species syntenic information . visualization of cross - species information in polybrowse : the genomic region shown in figure 1 is displayed herein to illustrate the syntenic capability . in this case , the g - quadruplex forming repeat track shows the locations of these features in the region near the beginning of the myc gene . the additional track illustrating the liftover1k blocks is also reported . in that track , when the user moves the cursor over the objects , a popup window appears that contains links ( a ) to the syntenic locations in the other available species . ( b e ) results upon clicking on the links for chimp , macaque , mouse and dog , respectively . currently , the liftover feature only allows moving from the human to the other species but not among the non - human species . ( a ) polybrowse view of g - quadruplex forming repeats in the myc gene with species syntenic information . visualization of cross - species information in polybrowse : the genomic region shown in figure 1 is displayed herein to illustrate the syntenic capability . in this case , the g - quadruplex forming repeat track shows the locations of these features in the region near the beginning of the myc gene . when the user moves the cursor over the objects , a popup window appears that contains links ( a ) to the syntenic locations in the other available species . ( b e ) results upon clicking on the links for chimp , macaque , mouse and dog , respectively . currently , the liftover feature only allows moving from the human to the other species but not among the non - human species . the computed non - b dna forming elements are likely to be under - represented in our reference genome as their underlying repeats may be polymorphic among individuals . because this type of information may be critical in the context of gene regulation or predisposition to disease ( 48 ) , we used a specific parser to scan both the reference human genome as well as additional sequence sources , such as trace reads from the trace archive ( http://www.ncbi.nlm.nih.gov/traces/trace.cgi ) and contigs ( http://www.ncbi.nlm.nih.gov/projects/wgs/wgsprojectlist.cgi ) from personal genome projects ( 49 ) , for matches to the non - b dna motifs . each match found in either the reference or alternate source is then scored for being polymorphic or not . of the sites identified as polymorphic , a second evaluation is made to determine whether the polymorphism would affect the motif underlying the putative non - b dna structure . the results of this scan are incorporated into the database as a series of separate tracks ( figure 3b , trace gplex tracks ) . additional information can be gathered by extending this type of analysis to sequence alignments using closely related species . currently , only the g - quadruplex forming motif supports this type of query . figure 3.(a ) polybrowse view of some of the non - b dna features in the human myc gene . links to polybrowse and ucsc browsers : ( a ) display of polybrowse rendering the myc gene and its promoter with some of the non - b dna motifs tracks with the pupy [ poly(purinepyrimidine ) ] and polymorphism tracks turned on . several other tracks include features computed at the advanced biomedical computing center ( abcc ) , such as strs , base composition , physical dna characteristics , mapping , as well as the ncbi derived features , including genes , snps , cytogenic markers assembly information , repeatmasker elements , etc . ( b ) display of the myc gene in polybrowse showing some of the polymorphism information in non - b db . below the gene and mrna tracks , the searched motifs in the reference sequence are displayed ( teal ) . an additional track shows specific g - quadruplex motifs in which all of the observed trace files contained mutations disrupting the g - quadruplex motif . ( c ) display of the myc gene at the ucsc human genome browser ( grch37/hg19 assembly ) as linked to non - b db from the search shown in figure 2b . some of the non - b dna motifs from non - b db can be seen in the red rectangular box . ( a ) polybrowse view of some of the non - b dna features in the human myc gene . links to polybrowse and ucsc browsers : ( a ) display of polybrowse rendering the myc gene and its promoter with some of the non - b dna motifs tracks with the pupy [ poly(purinepyrimidine ) ] and polymorphism tracks turned on . several other tracks include features computed at the advanced biomedical computing center ( abcc ) , such as strs , base composition , physical dna characteristics , mapping , as well as the ncbi derived features , including genes , snps , cytogenic markers assembly information , repeatmasker elements , etc . ( b ) display of the myc gene in polybrowse showing some of the polymorphism information in non - b db . below the gene and mrna tracks , the searched motifs in the reference sequence are displayed ( teal ) . some of the predicted motifs are found only in the trace sequences . an additional track shows specific g - quadruplex motifs in which all of the observed trace files contained mutations disrupting the g - quadruplex motif . ( c ) display of the myc gene at the ucsc human genome browser ( grch37/hg19 assembly ) as linked to non - b db from the search shown in figure 2b . some of the non - b dna motifs from non - b db can be seen in the red rectangular box . in order to provide access to the back - end database , we have leveraged two existing tools from the bioinformatics community . the first is a bioperl ( 50 ) set of methods , which is used to query genome databases in various ways , such as by position , by class , or by attribute . this same set of utilities is used within the context of polybrowse ( 44 ) , so that visualization of the genomic features is made available . in addition to linking the outputs from the query tools to the browser for visualization , we also provide links allowing the returned data to be displayed in the familiar ucsc ( http://genome.ucsc.edu/ ) genome browser ( figure 3c ) , as well as links to our biodbnet database warehouse ( 51 ) , which contains gene - centric information derived from several sources , and additional links . herein , we present a database containing the locations of motifs predicted to adopt the most common non - b dna structures . the database can be used to browse specific genomic regions for the possible contribution of non - b dna - forming elements to inherent biological observations derived from the region . in addition to the locations of predicted motifs , the database also contains polymorphism information about each of the test sequences , as well as additional candidate sequences not present within the reference genomes . additional genomes are in the process of being added to the system and will continue to be updated and added as they become available . input from the community regarding the addition of other tracks , enhanced algorithms for the detection or scoring of the identified motifs or additional query tools are welcome and will be incorporated into the system as appropriate . further additions , such as a community - based curation capability and the addition of other validation information through literature mining approaches are also under consideration . we anticipate that significant improvements to our methods will be made in the future by incorporating energetic , and other secondary metrics , to the current predictive algorithms . although significant biological knowledge would be required , such as localized superhelical density , nucleosome positioning , etc . ( see above ) , the overall goal is to associate a likelihood index with each of the predicted locations for each of the non - b dna - forming classes . finally , as reliable methods are expected to be developed that identify genome - wide data on non - b dna structures in vivo and some of the biological parameters involved , the resulting data sets can be used to train the prediction tools , resulting in improved predictive capabilities for each type of non - b - forming classes . center for biomedical informatics and information technology ( cbiit)/cancer biomedical informatics grid ( cabig ) isrce yellow task # 09 - 260 to nci - frederick and national cancer institute / national institutes of health contract hhsn261200800001e ( to a.b . ) . funding for open access charge : national cancer institute / national institutes of health contract hhsn261200800001e .

although the capability of dna to form a variety of non - canonical ( non - b ) structures has long been recognized , the overall significance of these alternate conformations in biology has only recently become accepted en masse . in order to provide access to genome - wide locations of these classes of predicted structures , we have developed non - b db , a database integrating annotations and analysis of non - b dna - forming sequence motifs . the database provides the most complete list of alternative dna structure predictions available , including z - dna motifs , quadruplex - forming motifs , inverted repeats , mirror repeats and direct repeats and their associated subsets of cruciforms , triplex and slipped structures , respectively . the database also contains motifs predicted to form static dna bends , short tandem repeats and homo(purinepyrimidine ) tracts that have been associated with disease . the database has been built using the latest releases of the human , chimp , dog , macaque and mouse genomes , so that the results can be compared directly with other data sources . in order to make the data interpretable in a genomic context , features such as genes , single - nucleotide polymorphisms and repetitive elements ( sine , line , etc . ) have also been incorporated . the database is accessed through query pages that produce results with links to the ucsc browser and a gbrowse - based genomic viewer . it is freely accessible at http://nonb.abcc.ncifcrf.gov .

INTRODUCTION RESULTS Non-B DB versus existing databases Non-B DB: key features Cross-species comparisons Polymorphism analysis CONCLUSIONS FUNDING

PMC4804645

diabetic patients are more prone to death following myocardial infarction ( mi ) independent of coronary vessel patency or left ventricular function . infarct size is independently associated with mortality after mi , and it is this increase in infarct size that is believed to be the cause of increased mortality in diabetic patients . the stz diabetic rat exhibits increased infarct size after ligation of the coronary artery compared to nondiabetic controls . this increased necrotic area may reflect either a generalized decreased ability of the diabetic myocardium to heal or an increased susceptibility of the myocardium to injury . the gap junction ( gj ) allows for organized propagation of electrical signals and metabolic coupling between cells . disruption of gjs is known to be associated with pathological states including arrhythmias in the heart and wound healing disturbances in the skin . green , becker , and coworkers reported that knockdown of the gap junction protein connexin 43 ( cx43 ) by antisense rna sped wound closure and limited extension of skin burn injuries in rodent models [ 6 , 7 ] . cx43 is markedly upregulated in dermal tissues surrounding diabetic foot ulcers and in clinical trials treatment with the cx43 carboxyl - terminal mimetic peptide ct1 significantly accelerates the rate of closure of these pathologic , slow - healing wounds . here , we used stz diabetic mice in conjunction with a previously described cardiac cryoinjury model , enabling precise control of the initial magnitude of tissue damage to the ventricle . consistent with observations in skin , we find that , compared to controls , the extent of myocardial damage is significantly increased in stz diabetic mice in association with notable changes in the organization of cx43 gjs and levels of intercellular coupling within ventricular tissues . animal care was in accordance with institutional guidelines at the medical university of south carolina , charleston , sc , usa ( animal welfare assurance number : a4328 - 01 ) . diabetes was induced in 13-week - old c57/blk6 mice ( charles river ) by an ip injection of 65 mg / kg streptozotocin ( sigma ) ( stz ) in 0.1 m citrate buffer 1 time per day for 3 days . one week after induction , tail vein glucose was measured using a accu - chek active blood glucose meter ( roche ) and mice were considered diabetic if nonfasting glucose was measured to be greater than 250 mg / dl . animals injected with citrate buffer only , or not injected , served as controls . as an additional control , a group of stz diabetic mice were injected with 2 units of lantus insulin ( sanofi aventis ) on alternating days for a 46-week time course for cryoinjury studies or a 26-week time course for dye coupling studies . to determine insulin effects on injury , insulin was administered 15 minutes to 1 hour before cryoinjury . in all cases , immediately prior to procedures ( either sampling for immunoconfocal , cryoinjury , or cell coupling studies ) , blood glucose was recorded . prior to procedures , insulin - treated stz mice were given additional insulin ( up to 5 u ) until tail vein glucose was < 200 mg / dl . control mice + insulin were given up to 5 u until tail vein glucose was < 100 mg / dl . control mice , not given stz , were only utilized if tail vein glucose was < 250 mg / dl . non - insulin - treated diabetic mice were excluded if tail vein glucose was < 250 mg / dl . after 46 weeks of streptozotocin - induced diabetes , mice exhibited significantly less weight gain and significantly higher tail vein blood glucose and appeared grossly ill . after 46 weeks of diabetes , mice underwent a survival surgery to induce nontransmural myocardial cryoinjury using protocols adapted from o'quinn and others . these mice were prepared for surgery by removing hair on the ventral surface with an electric clipper and disinfecting the area with betadine antiseptic solution . under anesthesia ( ketamine , 100 mg / kg , acepromazine , 3 mg / kg , ip , or 5% isofluorane ) , mice were placed in the supine position and the trachea was intubated with a 22-gauge angiocath over a blunt introducer . the mice were ventilated with 2% isoflurane in o2 at a tidal volume of 250 l and 150 cycles / minute . at this point , the animals received a subcutaneous injection of the analgesic carprofen ( 4 mg / kg ) to prevent postoperative pain . maintaining sterile technique , left thoracotomy was performed in the fourth intercostal space . all muscles overlying the intercostal space were dissected free and retracted with 5 - 0 silk threads . only the intercostal muscles were transected . after opening the pericardium , a prechilled cryoprobe ( ac3 : brymill cryogenics with a 3 mm flat surface cryoprobe ) the incision was closed in layers using 6 - 0 silk and the skin was sealed with dermabond veterinary adhesive . the mice were placed on ambient oxygen and allowed to recover until normal respiration was obtained . the animals received one additional carprofen injection ( q 24 hr ) before being returned to the housing facility . approximately 15 minutes before sacrifice , mice were administered 1000 units of heparin via ip injection . in the instance of respiratory distress ( shallow , abnormal breathing ) , for the assessment of injury size , hearts were apically perfused with pbs followed by 1% 2,3,5-triphenyltetrazolium chloride ( ttc : sigma ) in pbs for 5 minutes . the heart was removed from the animal and placed in a 37c pbs 1% ttc bath for 10 minutes followed by 4% paraformaldehyde overnight . epicardial surface area was calculated from images taken using leica mz fliii on digital images using imagej ( nih ) . as an additional measure of injury size , cryoinjured hearts were sagittally hemisectioned along the base - apex midline of the injury and frozen in optimal tissue cutting media cooled with liquid nitrogen . sections were fixed in 1% paraformaldehyde for 10 minutes and postfixed in an ice - cold 2 : 1 ethanol - acetic acid solution . specimens were stained with an apoptag fluorescein in situ apoptosis kit ( millipore , s7110 ) . it is more commonly known as terminal deoxynucleotidyl transferase mediated dutp nick end labeling ( tunel ) . sections were visualized using an epifluorescent microscope ( leica : dmlb ) equipped with 10x objective and a color chilled 3ccd camera ( hamamatsu ) . tunel staining was measured using imagej to measure the depth of the entire left ventricle visible by autofluorescence and the depth of tunel positive nuclei . this approach was based on lucifer yellow dye spread , a well - established method of assaying gj coupling in cultured cells , and further inspired by in vivo adaption of this method for study of skin wound healing in diabetic rats . mice were anesthetized with isofluorane and ventilated as described above . the thorax was opened and the heart was rapidly excised and washed quickly in 37c oxygenated tyrodes solution ( 126.5 mm nacl , 1.2 mm mgso4 , 1.2 mm kh2po4 , 14.5 mm nahco3 , 2.6 mm kcl , 20 mm cacl , and 22.1 mm glucose ) . the apex was transversely hemisectioned with an uncoated razor blade ( gem 62 - 0167 ) and snap frozen in liquid nitrogen for western blot analysis . the remaining freshly cut surface of the base was blotted dry with a kim wipe and the left ventricular region was placed directly on a 5 l drop of dye solution on a clean silguard coated surface . the dye solution was prepared fresh and contained 1% lucifer yellow ( sigma , l0259 - 25 mg ) and 10% alexa 647 dextran ( invitrogen , d22914 ) dissolved in pbs ( sigma , p4417 ) . the heart was removed from the dye after precisely 1 minute and washed quickly in 200 ml of pbs before being fixed in 4% paraformaldehyde for at least 10 minutes . after fixation , the hearts were washed twice in pbs and sagittally sliced along the left ventricular wall to expose the region of the heart through which dye had spread . the freshly cut edges were splayed apart on a glass slide and sandwiched below a coverslip . the coverslip was attached to the slide with wax on three sides forming a chamber around the specimen . the chamber was filled with flurogel - mount with tris buffer ( electron microscopy science , 17985 - 10 ) as an antifade reagent , and the 4th side of the chamber was then covered with wax to seal the specimen under the glass coverslip within the chamber . two two - channel single optical sections were recorded per heart in the alexa 488 and alexa 633 channels . the hearts were homogenized and lysed in 50 mm tris - hcl 1% np-40 , 150 mm nacl , 2 mm egta , 0.05 m naf , 100 um navo4 pmsf , and complete protease inhibitors . the samples were syringed thrice using a 22-gauge needle over ice and incubated at 4c with rotation for 30 minutes . protein concentration was determined using a bca kit and samples were diluted to 1.5 mg / ml . samples in 2x loading buffer were resolved in parallel on 12.5% tris - hcl gels ( criteron xt 345 - 0014 , bio - rad ) in 1x tris - glycine running buffer ( bio - rad ) . resolved proteins were transferred to immobilon pvdf at 15 v for 25 min ( 15 mm tris , 192 mm glycine , 10% vol / vol methanol , and 0.01% sds ) . the membranes were blocked in 5% nonfat dry milk / tbs - tween-20 and probed with primary antibodies . subsequently , blots were probed with goat anti - rabbit alkaline phosphatase secondary antibody ( 1 : 10,000 ; 4010 - 04 ; southern biotechnology ) . primary antibodies for cx43 blotting included s368 cx43 ( rabbit , cell signalling , 1 : 1000 ) , s262 cx43 ( rabbit , santa cruz , 1 : 500 ) , and total cx43 ( rabbit , sigma , 1 : 2000 ) . statistics and means were compared using student 's t - test ; p values < 0.05 were rejected as not significant . when appropriate , analysis of variance with posttesting was used for multiple comparisons . representative images of control ( figure 1(a ) ) and stz diabetic cryoinjury ( figure 1(b ) ) , as well as quantification of epicardial injury sizes ( figures 1(e)1(h ) ) , are shown in figure 1 . diabetic mice given insulin every other day and a bolus 1545 minutes before cryoinjury served as an additional control . these mice did not exhibit significantly larger injuries than nondiabetic mice , suggesting a protective effect of insulin in inhibiting injury spread in these hearts ( figure 1(e ) ) . an injury border zone was observed at the periphery of the central epicardial injury that demonstrated incomplete ttc staining ( figures 1(a ) and 1(b ) ) . the pink color observed in this region suggested the presence of both viable and nonviable tissue in this zone . interestingly , there was a trend towards a decrease in border zone size in the diabetic hearts ( figure 1(g ) ) , although this relationship was not significant ( p = 0.13 ) . tunel staining of cryoinjuries was performed to measure the area of apoptotic tissue ( figures 1(c ) and 1(d ) ) . however , tunel labelling of 48-hour cryoinjuries stained the entire injury ( figures 1(c ) and 1(d ) ) , making it difficult to discriminate between truly apoptotic cells and cells necrotic from the cryoinfarction . an interesting trend was observed where the depth of tunel labelling appeared greater in the diabetic hearts relative to controls ( figure 1(h ) ) ; however , this relationship was not significant ( n = 3 per group , p = 0.3 ) . as lack of insulin was associated with increased cryoinjury size observed in the diabetic mouse , we measured cryoinjury size in the control mice given insulin to induce hypoglycemia ( glucose < 100 mg / dl ) . while there was a trend towards decreasing injury size in the insulin - treated mice ( figure 2 ) , we detected no significant further reduction in injury size in the control mice given insulin compared to untreated control mice ( p = 0.3 ) . because insulin reduces cryoinjury spread in the diabetic heart , we next sought to determine whether intercellular coupling by gjs was associated with the increase in injury size in this model . we measured intercellular coupling by exposing freshly cut hearts to a solution containing the gj - permeant dye , lucifer yellow , and the gj impermeant dye , alexa 647 dextran . control hearts and diabetic hearts exhibited similar distances of lucifer yellow dye spread , which was significantly reduced in the presence of the gj uncoupler , heptanol . treatment of control or stz mice with insulin over the course of 1 week and provision of a bolus dose prior to cardiac excision to induce hypoglycemia ( glucose < 100 mg / dl ) resulted in a significant decrease in lucifer yellow dye spread throughout the ventricle ( p < 0.03 ) , as assessed by measuring the length of lucifer yellow dye spread from the site of furthest alexa 647 spread ( figure 3 ) . specifically , lampe and coworkers demonstrated that phosphorylation of cx43 at serine at its 368th amino acid residue ( serine 368 ) resulted in a 2-fold reduction of channel conductance . because decreased gj communication was detected in response to insulin , we sought to determine whether serine 368 phosphorylation was increased in the insulin - treated mouse heart . the apices of the insulin - treated hearts were homogenized and western blotted for phosphorylated cx43 p368 and total cx43 . a significant increase in p368 cx43/total cx43 in insulin - treated mouse apices was found ( p < 0.05 ) ( figure 4 ) . a similar increase in serine 368 levels was observed in diabetic mice given insulin ; however , insulin - nave diabetic mice demonstrated no detectable change in serine 368 levels ( data not shown ) . previous reports have indicated that serine 368 and serine 262 phosphorylation are both mediated by protein kinase c. phosphorylation at these residues is associated with a cardiac injury - resistant state . given that insulin increases serine 368 phosphorylation , we sought to determine whether insulin and/or diabetes exerted similar effects on serine 262 phosphorylation . in contrast to previous reports in nondiabetic animals , western blotting of serine 262 phosphorylated cx43 was substantially enhanced in the apices of stz diabetic mice ( figure 5(a ) , p < 0.05 ) . administration of insulin reduced serine 262 phosphorylation in these animals , suggesting that the increase was due to the hypoinsulinemia of diabetes and not an effect of the streptozotocin ( figure 5(b ) , left ) . a linear regression analysis of p262 cx43 versus tail vein glucose demonstrated a significant correlation between serine 262/total cx43 levels and blood glucose ( r = 0.516 , p = 0.0325 ) ( figure 5(b ) , right ) . immunofluorescence analysis of stz diabetic and control injured hearts confirmed an increase in serine 262 phosphorylated cx43 immunolabeling in these hearts ( figure 5(c ) ) . here , we report a number of novel findings : ( 1 ) stz diabetes increases the size of an epicardial cryoinfarction 48 hours after injury , in association with a trend in levels of gj - mediated intercellular coupling . the increased injury size can be effectively rescued by insulin administration , suggesting that the cause of the increased injury size is due to hypoinsulinemia . ( 2 ) insulin administration also reduces intercellular coupling in both diabetic and nondiabetic animals , suggesting that insulin induces uncoupling of gjs . ( 3 ) insulin administration is associated with increased levels of serine 368 phosphorylated cx43 . serine 368 is a residue known to be associated with reduced gj coupling and , consistent with the results presented herein , its phosphorylation in response to insulin has recently been reported in vitro and thus far has not been reported in vivo . our observation that insulin reduces cryoinjury size in stz diabetic mice is consistent with findings in other models of cardiac injury . marfella and colleagues demonstrated that infarct size in the stz diabetic mouse is increased in response to ischemia - reperfusion injury . but the clinical evidence is clear in that the diabetic heart is particularly sensitive to injury and that hyperglycemia , regardless of whether or not a diagnosis of diabetes has been made , is predictive of cardiac death . mri quantification studies of infarct size in patients with diabetes or dysglycemia presenting with acute myocardial infarction ( mi ) demonstrated that blood glucose levels were highly predictive of infarct size . it is long established that diabetic patients are at higher risk of death after a mi . it follows that the goal for treating dysglycemic patients in the setting of mi should be to reduce infarct size . we demonstrate that insulin - induced hypoglycemia significantly reduced gj - mediated intercellular transfer of lucifer yellow . becker 's group reported that cx43 was aberrantly upregulated in the dermis of diabetic skin and that gj coupling in the diabetic skin was increased . treatment of diabetic skin with cx43 antisense oligodeoxynucleotide reduced gj dye spread and increased the rate of reepithelialization of these wounds . in contrast to the aforementioned findings in diabetic skin , we detected no increase in cell coupling in the setting of diabetic heart ( figure 3 ) . the heart expresses large amounts of cx43 [ 1922 ] , compared to the dermis of skin . it may be that the modest increases in coupling that have been reported from studies of diabetic dermal tissues are not observable in ventricular myocardium of stz rodents , owing to the high reserve of cx43-mediated coupling occurring between myocardial cells . also , consistent with our findings using chemical cell - cell coupling assays , nygren and colleagues detected no discernable difference in electrical coupling by cardiac gjs in the stz diabetic rat heart . in the aforementioned study , conduction differences between diabetic and control hearts were only observed in the presence of gj uncoupler . the resolution of our lucifer yellow dye spread assay is likely not sensitive enough to detect this minor difference in coupling . the finding that insulin reduces gj coupling in the rodent heart is consistent with reports by homma and coworkers that demonstrated that insulin induces gj channel closure in xenopus oocytes . the effect of insulin on channel closure could be eliminated with the deletion of the ct of cx43 at amino acid 258 and restored by separately expressing portions of the cx43 ct . importantly , homma and coworkers reported that deletion of amino acids 261280 prevented insulin - induced closure of gjs in this model and coexpression of this sequence was sufficient to restore channel sensitivity to insulin . here , we report that phosphorylation of cx43 at serine 262 in the mouse heart correlates directly with tail vein glucose . taken together with the report of homma et al . , one possibility is that insulin - induced closure of gjs involves this residue . further supporting a role for serine 262 in regulating cell - cell dye propagation , doble and others demonstrated that an alanine mutation of serine 262 of cx43 resulted in increased dye propagation in cultured rat cardiomyocytes . mutation of other residues , including serine 368 , has been reported to prevent gj closure in response to pkc . in diabetes , pkc- expression is elevated and cx43 phosphorylation by pkc- is increased , consistent with the increase in serine 262 phosphorylation that we observe here . interestingly , we found that insulin administration increased levels of cx43 serine 368 phosphorylation in the mouse heart . homma and coworkers were unable to restore the effects of insulin on gj closure with a peptide construct containing serine 368 in xenopus oocytes . differential effects of insulin in xenopus oocytes and mammalian cardiomyocytes may explain these differing results . insulin translocates the epsilon isoform of protein kinase c ( pkc- ) from the cytosol to the membrane of myocytes in vitro . the time course is consistent with our observation of phosphorylation of cx43 in response to insulin administration in mouse hearts that received insulin immediately prior to sacrifice . isoforms of pkc are known to interact with cx43 at its ct and specifically phosphorylate serines 368 and 262 in mammalian tissues [ 25 , 28 ] . additionally , serine 368 phosphorylation is associated with a reduction in channel conductance and single channel permeability , results in agreement with our finding of reduced gj - mediated coupling in the mouse heart . the above points being raised , there are caveats to interpretation that should be made . here , we demonstrate that cryoinfarction produces larger injuries in diabetic mouse hearts , and , in turn , these are reduced by insulin pretreatment . additionally , we demonstrate that insulin administration increases serine 368 phosphorylation and this is associated with reduced cell coupling . however , it is well established that the effects of insulin are complex and multiform . insulin is an anabolic hormone involved in numerous signaling pathways and most importantly it regulates metabolic processes in the cell . insulin and igf effectors have been shown to enhance mitochondrial metabolism and be cardioprotective in the setting of cardiac failure . it is possible that the increased injury size found in stz diabetic mice is partially attributable to mitochondrial dysfunction that is acutely recovered by insulin treatment . additionally , because of multiple anabolic effects of insulin on protein expression , there is concern that insulin administration over the course of the week prior to sacrifice may have exhibited effects on cx43 expression in this setting . previous reports have documented that knockdown of greater than 95% of cx43 is required prior to observance of conduction slowing , which was not observed here . we observed no clear difference in cx43 expression level ; however , further work is needed to assess the effect of insulin administration on cx43 expression . further work is also needed to address how serine 368 phosphorylation of cx43 is regulated and to define its role in the normal and diabetic heart in response to insulin and other cardiac modulating agents . early reports by lin and others demonstrated an increase in cx43 phosphorylation ( as identified by different molecular weight bands via immunoblotting ) in the diabetic rodent . we expand on these findings here by characterizing phosphospecific isoforms of cx43 in the diabetic heart . we found that cx43 phosphorylated at serine 368 was not increased in the diabetic heart but in fact decreased and determined that phosphorylation at this site was increased by insulin administration . the additional site , serine 262 which is also phosphorylated by pkc- , exhibits increased phosphorylation in diabetes and may be the residue responsible for the shift in cx43 molecular mass noted in the report of lin et al . . one tempting hypothesis that stems from our findings is that differential phosphorylation of the cx43 ct regulates its role in the response to injury . in diabetes , the disparity in phosphorylation observed in diabetes suggests that conformational changes within the cx43 ct , or its interaction with other binding partners , may block the ability of pkc- to efficiently phosphorylate cx43 at s368 in the setting of the non - insulin - treated diabetic heart . in this study , we demonstrated that an increased area of cell death occurs after cryoinjury in the diabetic mouse heart and that this increase was reduced by a preinjury treatment with insulin . insulin administration was associated with increased levels of serine 368 phosphorylation of cx43 and decreases in gj - mediated intercellular coupling . additionally , we reported that diabetes results in increases in serine 262 phosphorylation of cx43 . we conclude that insulin is cardioprotective against cryoinjury in our mouse model and that this protection is associated with decreased cell - to - cell coupling . these findings suggest that glycemic control prior to a planned cardiac injury ( i.e. , cardiac surgery ) may assist in the preservation of myocardium . furthermore , modulation of gap junction - mediated intercellular communication may be a potential therapeutic target for myocardial injury preservation strategies . this being said , further work in humans is needed to evaluate the safety and efficacy of this approach .

diabetic patients develop larger myocardial infarctions and have an increased risk of death following a heart attack . the poor response to myocardial injury in the diabetic heart is likely related to the many metabolic derangements from diabetes that create a poor substrate in general for wound healing , response to injury and infection . studies in rodents have implicated a role for the gap junction protein connexin 43 ( cx43 ) in regulating the injury response in diabetic skin wounds . in this study , we sought to determine whether diabetes alters cx43 molecular interactions or intracellular communication in the cryoinjured stz type i diabetic mouse heart . we found that epicardial cryoinjury size is increased in diabetic mice and this increase is prevented by preinjury insulin administration . consistent with these findings , we found that intercellular coupling via gap junctions is decreased after insulin administration in diabetic and nondiabetic mice . this decrease in coupling is associated with a concomitant increase in phosphorylation of cx43 at serine 368 , a residue known to decrease channel conductance . taken together , our results suggest that insulin regulates both gap junction - mediated intercellular communication and injury propagation in the mouse heart .

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

PMC3015816

when compared with abdominal hysterectomy ( ah ) and laparoscopic - assisted vaginal hysterectomies ( lavh ) , tlh has been reported to result in shorter procedure durations , lower blood losses , and shorter hospital stays . typically , vaginal hysterectomy ( vh ) and lavh are performed in patients with at least moderate prolapse usually associated with parity , but some of these patients may later develop vaginal prolapse or incontinence . one recent report describes recurrence of prolapse in 12% of patients having a vaginal hysterectomy with vaginal vault suspension compared with 4% having a laparoscopic uterosacral ligament uterine suspension . among nulliparous patients , the vaginal dissection may be difficult due to absence of prolapse and small vaginal capacity and may result in more complications . tlh may offer a minimal blood loss , short hospital stay , and be practicable in most women with minimal risk of complications , but randomized trials are lacking . a recent cochrane meta - analysis of hysterectomy approaches reviewed 24 randomized trials of 3643 women , but only 2 trials with 71 women were identified for type vii tlh . very few contemporary newly trained gynecologists will have sufficient expertise and confidence to tackle tlh , which requires the highest level of surgical skill . they concluded that [ t]he newest approach to hysterectomy ( tlh ) should be further evaluated . it is speculated that if a tlh technique used familiar open technique standards and was observed to be safe in a large retrospective series , potentially more surgeons would learn and safely perform the technique , and randomized controlled trials could be designed to compare the various routes . in this article , technique and complications are reviewed and analyzed in great detail so that surgeons learning and newly performing tlh may avoid complications , and subsequent randomized trials could be planned . all tlh cases were identified from computerized billing lists by using the current procedure terminology codes for all types of hysterectomy . approval by the investigation review board at sequoia hospital in redwood city , california , has been maintained with yearly updates . all consecutive , eligible patients were offered simple tlh for every benign gynecologic indication , stage ia1 ( < 37 mm invasion ) cervical cancer , occult ovarian cancer , and clinical stage iia or less endometrial cancer . patients were ineligible if they had documented severe abdominal adhesions from a prior operative report , or endometrial cancer in a uterus too large for intact removal through the vagina . every surgery was performed by the author ( kao'h ) from september 5 , 1996 to april 1 , 2006 , at 4 california hospitals , assisted by a categorical obstetrics and gynecology resident , a gynecologist , or a general surgeon . for clarification in this article , the technique used for this type vii tlh the patient is in modified lithotomy position with hips extended , in a 40-degree angle trendelenburg position . shoulder bolsters prevent slippage up the table , and the arms are padded and tucked by the side . after the examination with the patient anesthetized , a humi uterine manipulator ( cooper surgical , inc . trumbull , ct , usa ) is inserted , unless the patient has endometrial cancer , in which case the manipulator is inserted after laparoscopic washings and bipolar cautery occlusion of the proximal fallopian tubes . after visual inspection of the ureter at the pelvic brim , the infundibulopelvic ligament , or , utero - ovarian ligament is coagulated and incised with an ultrasonic scalpel ( ethicon endo - surgery , cincinnati , oh , usa ) . immediately after the round ligament is incised , the uterus , on the uterine manipulator , is pushed cephalad to recreate the traction - counter - traction a bladder flap is incised and the anterior cervical fascia is exposed with blunt dissection off of the cervix broadly below the cervicovaginal margin . the uterine arteries are coagulated with a bipolar cautery at the mid lower cervical length , and then incised with the ultrasonic scalpel . the uterine arteries are pushed downward to expose the cardinal ligament fibers attaching the arteries to the cylindrical cervical fascia ; then the cardinal fibers are incised posteriorly to the uterosacral ligaments , and inferiorly , identifying the cervicovaginal margin as the lowest limit of dissection . the cervicovaginal margin is laparoscopically palpated with the laparoscopic instruments by using visual and manual cues to delineate the posterior margin , the lateral edges , and the anterior margin of the cervical stroma ( figure 1 ) . typically , the cervical stroma is firm and moves as a solid object , and the vaginal wall is pliant and dimples with pressure . if the cervicovaginal margin is not obvious , a thin malleable ribbon is inserted into the anterior vagina and elevated , identifying the anterior - most cervicovaginal margin . then the ultrasonic scalpel incises into the vagina at the precise margin of the cervix and vagina ( figure 2 ) . when the pneumoperitoneum is lost , the uterine manipulator is removed and a surgical glove containing 2 fluffed 44 gauze sponges is inserted to obturate the lower vagina . two semb toothed cupped graspers ( # a5261 , olympus surgical , orangeburg , ny , usa ) are used to expose the cervicovaginal margin for ultrasonic incision around the cervix . a tenaculum is inserted through the vagina ( under or beside the glove ) to grasp the cervix and remove the uterus . when the uterus is large , but only in the absence of endometrial cancer , it is morcellated transvaginally . any pelvic mass is placed intact in a lapsac surgical tissue pouch ( # 054100 , cook urological , spencer , in , usa ) for frozen section . the edges of the pouch are brought out through the introitus , and a speculum is inserted into the pouch , allowing puncture or morcellation and removal of the mass with no intraperitoneal spillage . the vaginal apex is closed with # 1 polygalactic acid jk10 ( ethicon endo - surgery , cincinnati , oh , usa ) suture in 3 or more figure - of - eight ( technically spiral ) sutures with semb graspers and a wolfe needle driver ( # 8393.502 , richard wolf surgical instruments , vernon hills , il , usa ) , fixing the vaginal angle to the uterosacral ligaments for suspension . anova , chi - square , and spearman and pearson correlation techniques were used with significance set at p<0.05 . the right lower uterine segment is seen with suction irrigator delineating the posterior cervicovaginal margin . after incising the cardinal ligament , the pliant vaginal wall is palpated laterally , anteriorly , and posteriorly to confirm the location of the cervicovaginal margin . three had massive myomata in the lower uterine segment obstructing laparoscopic ligation of the uterine arteries . the fourth patient was converted to laparotomy to explore a limited retroperitoneal bleeding site that was identified as a likely trocar injury . the fifth patient had persistent bleeding of the left infundibulopelvic vessels that had retracted into the retroperitoneum . preoperative diagnoses additional procedures performed with total laparoscopic hysterectomy * some patients had more than one procedure . all cases of malignancy were managed with strict standards of oncological principles : pelvic masses were removed intact in a ripstop nylon bag . the typical patient in this series had a mean age of 50.1 years ( sd=11.0 ; range , 21 to 90 ) . the average parity was 1.3 ( sd=1.3 ; range , 0 to 9 ) with 38.2% ( n=315 ) of these women being nulliparous . their mean body mass index ( bmi ) was 27.8 kg / m ( sd=7 ; range , 16 to 71 ) with 57% ( n=468 ) of the women being in the overweight or higher bmi range ( > 24.9kg / m ) . surgical duration was abstracted from operating room records and included all gynecologic procedures and incidental appendectomy . patients requiring staging lymphadenectomy and omentectomy for malignancy , or other general surgical procedure such as cholecystectomy were not included in the duration analysis . the mean surgery duration was 130 minutes ( sd=56 ; range , 28 to 355 ) . over one fifth ( 23.7% ) of patients ' surgeries were completed in 90 minutes or less . neither age ( r=0.025 , p=0.513 ) , body mass index ( r=0.060 , p=0.115 ) , nor parity ( t=1.896 , p=0.058 ) impacted duration , but the increasing number of cases performed by the surgeon reduced duration ( r=0.448 , p<0.001 ) . blood loss was estimated by examining the contents in the canister before irrigation , or by surgeon and anesthesiologist agreement . the average blood loss was 130 ml ( sd=178 ; range , 0 to 1200 , median 75 ) with 16% of patients ( n=132 ) losing less than an estimated 10ml of blood . this estimate is made when there is virtually no observable blood lost during the procedure , little or none observed in the pelvic cul de sac at the end of the procedure , and little or none lost from the laparoscopic incisions . more than half of the patients lost less than 50 ml of blood . while age ( r = 0.018 , p=0.633 ) and bmi ( r=0.050 , p=0.184 ) did not correlate with blood loss , lower parity ( t = 2.91 , p=0.004 ) and increasing experience ( r=0.247 , p<0.001 ) correlated with reduced blood loss . transfusions , mostly related to complications , were needed in 3.0% of women ( 11.0 vs. 1.7% , p=0.0001 ) , especially a reoperative complication ( 13 vs. 2.4% , age was not significantly related to having a transfusion ( t=0.09 , p=0.93 ) , nor was bmi ( t=0.7109 , p=0.48 ) . however , those requiring transfusions had longer surgery ( t=3.50 , p=0.000 ) and more days in the hospital ( t=2.35 , p=0.028 ) . the median uterine weight was 160 , and the mean was 239 g , ( sd = 291 ; range , 50 to 3131 ) . of the 632 with recorded uterine weights , 16% were between 250 grams and 499 g , and 12% were between 500 grams and 3131 g. bmi did not impact uterine weight ( r=0.056 , p=0.156 ) ; however , increasing age correlated with smaller uteri ( r = 0.113 , p=0.004 ) . having a larger uterus correlated with increased surgical time ( r=0.268 , p<0.001 ) and more blood loss ( r=0.347 , p<0.001 ) . seven nulliparous , obese patients with bmi ranging from 31 to 46 , and massive uteri , had a tlh followed by a planned 6-cm pfannenstiel incision used only for evacuation of the disconnected uterine tissue , weighing 1047 g to 3131 g. only evacuation of tissue was performed through the pfannenstiel incisions . the pfannenstiel incisions were immediately closed , and the remainder of the case , including suture of the vaginotomy , was completed laparoscopically . had any portion of these cases required laparotomy for completion of the hysterectomy / oophorectomy dissection , these cases would have been classified as converted or as a laparoscopic - assisted abdominal hysterectomy . the average hospital stay was 1.37 days ( sd=0.97 ; range , 0 to 13 , median , 1 day ) . duration of hospital stay was recorded only by days , not hours ; however , 73% of patients left the hospital by lunchtime the next day . longer stays were not associated with age , bmi , parity , or uterine size , but did correlate with longer surgical times ( r=0.347 , p<0.001 ) and more blood loss ( r=0.346 , p<0.001 ) . the length of hospital stay decreased with the increasing number of cases performed by the surgeon ( r=0.452 , p<0.001 ) . complications occurred in 83 patients ( 10% ) , and of those complications about half required reoperation ( 39 patients , 4.7% ; table 3 ) . urologic complications occurred in 23 ( 2.8% ) patients and resulted in reoperation in about half ( 1.4% ) . complications were more likely to occur in women with a lower bmi ( or 0.95 ; 95% ci=0.90 to 0.99 ) , with a high surgical blood loss ( or 1.002 ; 95% ci=1.001 to 1.003 ) resulting in longer operating times ( or 1.007 ; 95% ci=1.003 to 1.012 ) and a longer length of stay in the hospital ( or 1.78 ; 95% ci=1.39 to 2.28 ) . the demographics , diagnoses , and surgicopathological data reflect a broad diversity of consecutive patients and demonstrate a broad utility for tlh . senior age and high bmi are not contraindications to tlh . because type vii tlh does not depend on vaginal descensus , capacity , or laxity , minimally invasive hysterectomy is available to more women , including the nulliparous and obese . with a 38% nulliparity rate , few of these patients would have qualified for a vh or an lavh . agostini et al report a high rate of successful vaginal hysterectomy in their 52 nulliparous patients , but their complication rate was 13.46% , higher than our total complication rate for this subset ( 10.82% ) , and much higher than our reoperative rate ( 4.5% ) in this subset . their hemorrhage rate was 7.69% , which was much higher than our transfusion rate of 3.2% . this report responds to their conclusion that laparoscopic assistance for nulliparous women having hysterectomy needed further investigation . their findings underscore the benefit of performing the entire surgery laparoscopically rather than struggling through a narrow vagina to reach a cervix with no descensus . it is unfortunate that operating time for tlh only was not recorded in the operating room records . the mean operating time of 132 minutes is thus an overestimate of the time needed for tlh only . the duration decreased with surgeon 's experience , with the last 100 cases taking 99 minutes or less . in controlled trials , type vii tlh has been observed to take the same or slightly more time than ah , similar or shorter time than lavh , and 53 minutes longer than vh . it is a weakness of this study that blood loss was not measured in a more precise fashion . in randomized trials , type vii tlh confers less blood loss than ah and lavh do , and confers a similar blood loss as that of vh . precise assessment of patient hospital stays would have required calculation of the hours from the end of surgery to the hour of discharge . patients stayed in the hospital 2 days ; however , in the second half of the series , it became routine to discharge patients on postoperative day one . this has caused no problems , and no readmissions have been observed for an extra day of observation . randomized trials reveal that type vii tlh confers a shorter hospital stay than do ah and lavh , and a similar stay as that of vh . uterine size did not correlate with increasing likelihood of complication ( p=0.6705 ) , urologic complication ( p=0.5782 ) , or conversion to laparotomy , as others have seen . lower uterine segment fibroids in 3 cases precluded access to the uterine arteries necessitating conversion to laparotomy for bleeding control . preoperative evaluation of the parametrium of patients with lower uterine segment fibroids can identify patients with an increased risk of laparotomy . laparoscopic myomectomy for optimal visualization of the cervical anatomy is used when lower uterine fibroids are large . the total complication rate of 9.8% and major complication rate of 4.5% in this series are comparable to rates in other tlh series ( table 3 ) . hoffman et al had a total and major complication rate of 10% and 5.6% , while heinberg et al had a major complication rate of 14.4% , and chapron reported complications in 10% . a complication rate of 13.4% has been observed for vh performed on nulliparous women , and 18% for vh after cesarean delivery . in randomized , controlled trials , type vii tlh conferred fewer complications , fewer wound infections , similar urologic complication rates , and was more cost effective than was ah , and resulted in comparable complications as lavh and vh . six categories of complications are analyzed for possible prevention strategies : urologic injuries , intestinal injuries , hemorrhagic events , infections , wound healing problems , and retained device complications . nineteen ( 2.3% ) patients had a urological injury , with half requiring reoperation : 3 cystoscopic ureteral stent placements , 4 ureteral reimplantations , and 2 laparotomies for closure of bladder fistula . tlh urological injury rates are reported at 2.5% to 3.4% and 2.2% in the first half of one series , decreasing to 0.9% in the second half . six injuries occurred during bladder flap dissections in patients with large lower uterine fibroids ( uterine weights all > 510 g ) and 3 with prior cesarean deliveries . one injury occurred during dissection of the anterior abdominal wall peritoneum to access the space of retzius for a burch procedure . to repair these , a running 3- 0 polygalactic acid suture in 2 layers it was successful in all but 2 patients who required laparotomy for successful repair after developing delayed breakdown of the laparoscopic cystotomy repair . when the margins of the bladder are not obvious before any dissection , inflating the bladder with a small amount of carbon dioxide can effectively delineate the edges . by clamping a hemostat on the foley catheter , attaching the insufflator tubing to it , and slowly opening the clamp while laparoscopically watching the bladder inflate with a small amount of carbon dioxide , patients typically have scarring on the upper portion of the anterior cervix and lower uterine segment , extending as low as the middle level of the cervix , but rarely ever lower . to dissect below this scar to unoperated vesicocervical fascia , the broad ligament adventitia is opened widely along the anterolateral aspect of the cervix until below the scar , where the smooth anterior white fascia of the cervix is easily separated from the bladder . then cutting ultrasonic dissection of the scar ureterotomy or ureteral transsection with immediate and successful repair occurred in 2 patients . in one case very early in the series ( case # 31 ) , in the other , redundant peritoneum was being trimmed after the completed hysterectomy for a massive , predominantly retroperitoneal fibroid uterus , without reconfirmation of the ureteral location . in both cases , a stent was inserted cystoscopically , and laparoscopically placed in the proximal portion of the ureter , with ureteroureterostomy by using 4 - 0 vicryl suture on the spatulated ends , and peritoneal closure without a drain . constant attention to the ureteral location would have prevented both injuries . eight ureteral fistulae developed postoperatively , most in the first third of the series , due to 2 sites of potential injury.it is well known that lack of recognition of the ureter in the lateral parametrial canal when ligating the uterine artery can result in ureteral injury 4 cm to 5 cm from the ureterovesical junction . an additional site of ureteral injury has come about solely through tlh due to the lack of precise recognition of the cervicovaginal margin and dissection too far down the vagina . dissection below the cervicovaginal margin can injure the ureter 2 cm to 3 cm from the ureterovesical junction . in open cases , surgeons have easily avoided this injury by carefully palpating the cervicovaginal margin between thumb and forefinger . surgeons keep the uterus on traction - counter - traction during laparotomy and feel the cervicovaginal margin repeatedly before incising into the vagina precisely at the cervicovaginal margin . the standard for laparoscopic surgeons must be the same : always know the exact site of the cervicovaginal margin by palpating the cervicovaginal margin with laparoscopic instruments to feel the precise change from the firmness of the cervical body to the pliancy of the vaginal wall , anteriorly , posteriorly , and laterally . when any of these landmarks are in question , a long right - angled heaney retractor or 1-inch ribbon is inserted to the anterior vagina and elevated , and the vaginotomy is performed safely onto the retractor 's edge at the anterior central cervicovaginal margin . cystoscopy after tlh should be performed in any case in which bladder or ureteral integrity is in question , to confirm closure of any repaired defect or to confirm patency of ureters after procedures , such as uterosacral ligament plications or burch colposuspensions . a 5-mm laparoscope can be used after instilling saline in the bladder with the suction irrigator . no indigo carmine is required to see the ureteral jets . using the irrigator and laparoscope gently as described three intestinal injuries occurred in the series ; only one was recognized immediately and repaired . one patient had slippage of the trocar outside of the abdominal cavity , with reinsertion performed without direct observation , through the bowel wall , requiring a figure - of - eight imbrication of both sites with 3- 0 polygalactic acid suture . the other 2 were occult injuries , presenting on days 7 and 29 , possibly from a lighted scope left in the abdomen touching the bowel , or inadvertent injury from ultrasonic scalpel dissection . three patients developed obstructive small - bowel adhesions to the vaginal apex , as evidenced by prolonged ileus and radiological demonstration of small - bowel obstruction at the vaginal apex . vaginal hemorrhage , retroperitoneal hemorrhage , and intraabdominal hemorrhage occurred in 11 ( 1.3% ) , 6 ( .8% ) , and 5 ( .7% ) patients , respectively , as observed in other series . of the 11 patients with vaginal hemorrhage , 5 occurred in the recovery room with immediate return to the operating room for additional suture placement vaginally . using at least 3 figure - of - eight ( technically spiral ) sutures along the vaginal apex seems essential , even when there is good hemostasis of the cuff edge . six patients developed brisk arteriolar cuff bleeding on days 7 through 10 , and 4 required sutures in the office . none of these 4 patients were diagnosed with a coagulopathy ; however , the suture material appeared dissolved in each . two patients had bleeding remote from the hospital and were observed in an emergency room without suture . of the 11 patients with abdominal or retroperitoneal hemorrhage , 6 were managed expectantly because they maintained normal vital signs or presented long after discharge with hematoma , which resolved . two had unexpectedly intense pain in the first 12 hours after surgery , which might have been an early warning symptom . no bleeding sites were identified in any of the reoperative cases , and none required laparotomy . nine patients ( 1% ) were diagnosed with pelvic cellulitis and received oral antibiotics with resolution of symptoms . five ( 0.4% ) patients were suspected of having pelvic abscess : 2 had 3.0-cm fluid collections with ct aspirate of clear sterile fluid . three had abscess : 2 were laparoscopically irrigated and drained , while one patient required laparotomy . four patients developed small - bowel herniation into a 5-mm trocar site , with 3 diagnosed after discharge from the hospital . each patient developed nausea and abdominal distension ; and ct was confirmatory in all cases . in each case , laparoscopic release resulted in recovery of bowel function after 3 days . herniation into a 5-mm trocar may occur with more degrees of instrument manipulation stretching the fascial incisions . five patients have had partial vaginal dehiscence , most after sexual penetration after the 6-week postoperative vaginal examination . one patient developed complete dehiscence of the vaginal apex 18 days after her surgery , and required operative repair . none of the other 4 patients required surgical repair , but all patients were advised to use a 1-cm thick foam ring around their partner 's penis during sexual activity to prevent their deepest penetration for the next 3 months . there have been no further incidents since warning all patients of the 1% chance of rupture with deep penetration after 6 weeks . an 8-mm segment of metal element from the bipolar cautery was retrieved laparoscopically from 1 patient in postoperative week 4 . most of the gynecological , hemorrhagic , infectious , and general surgical complications were addressed laparoscopically , vaginally , or locally . while all of the urological complications were treated by laparotomy except for cystoscopic stenting , this statistic will likely shift in time to laparoscopic repair . nineteen ( 2.3% ) patients had a urological injury , with half requiring reoperation : 3 cystoscopic ureteral stent placements , 4 ureteral reimplantations , and 2 laparotomies for closure of bladder fistula . tlh urological injury rates are reported at 2.5% to 3.4% and 2.2% in the first half of one series , decreasing to 0.9% in the second half . six injuries occurred during bladder flap dissections in patients with large lower uterine fibroids ( uterine weights all > 510 g ) and 3 with prior cesarean deliveries . one injury occurred during dissection of the anterior abdominal wall peritoneum to access the space of retzius for a burch procedure . to repair these , a running 3- 0 polygalactic acid suture in 2 layers it was successful in all but 2 patients who required laparotomy for successful repair after developing delayed breakdown of the laparoscopic cystotomy repair . when the margins of the bladder are not obvious before any dissection , inflating the bladder with a small amount of carbon dioxide can effectively delineate the edges . by clamping a hemostat on the foley catheter , attaching the insufflator tubing to it , and slowly opening the clamp while laparoscopically watching the bladder inflate with a small amount of carbon dioxide , , patients typically have scarring on the upper portion of the anterior cervix and lower uterine segment , extending as low as the middle level of the cervix , but rarely ever lower . to dissect below this scar to unoperated vesicocervical fascia , the broad ligament adventitia is opened widely along the anterolateral aspect of the cervix until below the scar , where the smooth anterior white fascia of the cervix is easily separated from the bladder . then cutting ultrasonic dissection of the scar ureterotomy or ureteral transsection with immediate and successful repair occurred in 2 patients . in one case very early in the series ( case # 31 ) , parametrial dissection landmarks were distorted by large lower uterine segment fibroids . in the other , redundant peritoneum was being trimmed after the completed hysterectomy for a massive , predominantly retroperitoneal fibroid uterus , without reconfirmation of the ureteral location . in both cases , a stent was inserted cystoscopically , and laparoscopically placed in the proximal portion of the ureter , with ureteroureterostomy by using 4 - 0 vicryl suture on the spatulated ends , and peritoneal closure without a drain . eight ureteral fistulae developed postoperatively , most in the first third of the series , due to 2 sites of potential injury.it is well known that lack of recognition of the ureter in the lateral parametrial canal when ligating the uterine artery can result in ureteral injury 4 cm to 5 cm from the ureterovesical junction . an additional site of ureteral injury has come about solely through tlh due to the lack of precise recognition of the cervicovaginal margin and dissection too far down the vagina . dissection below the cervicovaginal margin can injure the ureter 2 cm to 3 cm from the ureterovesical junction . in open cases , surgeons have easily avoided this injury by carefully palpating the cervicovaginal margin between thumb and forefinger . surgeons keep the uterus on traction - counter - traction during laparotomy and feel the cervicovaginal margin repeatedly before incising into the vagina precisely at the cervicovaginal margin . the standard for laparoscopic surgeons must be the same : always know the exact site of the cervicovaginal margin by palpating the cervicovaginal margin with laparoscopic instruments to feel the precise change from the firmness of the cervical body to the pliancy of the vaginal wall , anteriorly , posteriorly , and laterally . when any of these landmarks are in question , a long right - angled heaney retractor or 1-inch ribbon is inserted to the anterior vagina and elevated , and the vaginotomy is performed safely onto the retractor 's edge at the anterior central cervicovaginal margin . cystoscopy after tlh should be performed in any case in which bladder or ureteral integrity is in question , to confirm closure of any repaired defect or to confirm patency of ureters after procedures , such as uterosacral ligament plications or burch colposuspensions . a 5-mm laparoscope can be used after instilling saline in the bladder with the suction irrigator . no indigo carmine is required to see the ureteral jets . using the irrigator and laparoscope gently as described three intestinal injuries occurred in the series ; only one was recognized immediately and repaired . one patient had slippage of the trocar outside of the abdominal cavity , with reinsertion performed without direct observation , through the bowel wall , requiring a figure - of - eight imbrication of both sites with 3- 0 polygalactic acid suture . the other 2 were occult injuries , presenting on days 7 and 29 , possibly from a lighted scope left in the abdomen touching the bowel , or inadvertent injury from ultrasonic scalpel dissection . three patients developed obstructive small - bowel adhesions to the vaginal apex , as evidenced by prolonged ileus and radiological demonstration of small - bowel obstruction at the vaginal apex . vaginal hemorrhage , retroperitoneal hemorrhage , and intraabdominal hemorrhage occurred in 11 ( 1.3% ) , 6 ( .8% ) , and 5 ( .7% ) patients , respectively , as observed in other series . of the 11 patients with vaginal hemorrhage , 5 occurred in the recovery room with immediate return to the operating room for additional suture placement vaginally . using at least 3 figure - of - eight ( technically spiral ) sutures along the vaginal apex seems essential , even when there is good hemostasis of the cuff edge . six patients developed brisk arteriolar cuff bleeding on days 7 through 10 , and 4 required sutures in the office . none of these 4 patients were diagnosed with a coagulopathy ; however , the suture material appeared dissolved in each . two patients had bleeding remote from the hospital and were observed in an emergency room without suture . of the 11 patients with abdominal or retroperitoneal hemorrhage , 6 were managed expectantly because they maintained normal vital signs or presented long after discharge with hematoma , which resolved . two had unexpectedly intense pain in the first 12 hours after surgery , which might have been an early warning symptom . no bleeding sites were identified in any of the reoperative cases , and none required laparotomy . nine patients ( 1% ) were diagnosed with pelvic cellulitis and received oral antibiotics with resolution of symptoms . five ( 0.4% ) patients were suspected of having pelvic abscess : 2 had 3.0-cm fluid collections with ct aspirate of clear sterile fluid . three had abscess : 2 were laparoscopically irrigated and drained , while one patient required laparotomy . four patients developed small - bowel herniation into a 5-mm trocar site , with 3 diagnosed after discharge from the hospital . each patient developed nausea and abdominal distension ; and ct was confirmatory in all cases . in each case , laparoscopic release resulted in recovery of bowel function after 3 days . herniation into a 5-mm trocar may occur with more degrees of instrument manipulation stretching the fascial incisions . five patients have had partial vaginal dehiscence , most after sexual penetration after the 6-week postoperative vaginal examination . one patient developed complete dehiscence of the vaginal apex 18 days after her surgery , and required operative repair . none of the other 4 patients required surgical repair , but all patients were advised to use a 1-cm thick foam ring around their partner 's penis during sexual activity to prevent their deepest penetration for the next 3 months . there have been no further incidents since warning all patients of the 1% chance of rupture with deep penetration after 6 weeks . an 8-mm segment of metal element from the bipolar cautery was retrieved laparoscopically from 1 patient in postoperative week 4 . most of the gynecological , hemorrhagic , infectious , and general surgical complications were addressed laparoscopically , vaginally , or locally . while all of the urological complications were treated by laparotomy except for cystoscopic stenting , this statistic will likely shift in time to laparoscopic repair . this technique for type vii tlh appears safe and allows excellent access to the entire abdomen as needed in cancer surgeries , for patients with pelvic mass , endometriosis , pain , or adhesions with minimal morbidity . a total laparoscopic capability makes the benefits of a minimally invasive approach available to more women , including obese and nulliparous women . with an understanding of the complications from this technique , it is hoped that complications can be avoided and that more surgeons will safely learn tlh . experienced laparoscopic surgeons are urged to initiate needed randomized clinical trials of type vii tlh .

objective : this study analyses the technique and complications from total laparoscopic hysterectomy.methods:retrospective chart abstraction was performed on 830 consecutive patients operated on between 1996 and 2006 . demographic and surgical data were analyzed by anova , chi - square , and spearman and pearson correlation techniques were used with significance set at p<0.05.results : of 830 consecutive patients , 5 ( 0.6% ) were converted to laparotomy . patients had a mean age of 50 ( 11 ) years , a mean of 1.3 ( 1.3 ) pregnancies , and a mean bmi of 27.6 ( 6.8 ) kg / m2 . the mean surgical duration was 132 ( 55 ) minutes , with mean blood loss of 130 ( 189 ) ml and average hospital stay of 1.4 ( 0.9 ) days . duration of surgery , blood loss , and hospital stay all decreased with the surgeon 's increasing experience . reoperative complications occurred in 38 patients ( 4.7% ) . urologic injuries were observed in 23 patients ( 2.6% ) , with 9 ( 1.1% ) requiring reoperation.conclusions:this technique for tlh offers the benefits of minimally invasive surgery for patients needing hysterectomy , even those without vaginal capacity and uterine prolapse .

INTRODUCTION METHODS RESULTS DISCUSSION Urologic Injuries Intestinal Injuries Hemorrhagic Events Infections Wound Healing Problems Retained Device CONCLUSION

PMC4279973

bisphosphonates ( bps ) are used to treat osteoporosis , bone metastasis and other conditions involving fragile bone . oral bps are used primarily in the treatment of osteoporosis , and alendronate is one of the most potent antiosteoporotic agents known . as a side effect of using bps , bp - related osteonecrosis of the jaw ( bronj ) was reported in 2003 ; in the field of oral and maxillofacial surgery , it is one of the most difficult diseases to treat1 . alendronate pharmacologically inhibits farnesyl diphosphate synthase in the mevalonate pathway , which is essential for the prenylation of proteins in osteoclasts . this ultimately causes mechanical inhibition of osteoclast adhesion on the bone margin where absorption takes place through osteoclast apoptosis . the effect of bps on osteoclasts could also arise , at least in part , from modulation of the synthesis of resorptionpromoting or resorption - inhibiting factors by osteoblasts2 . osteoblast / stromal cells regulate osteoclastogenesis by producing proteins such as macrophage colony - stimulating factor ( m - csf ) , receptor activator of nuclear factor-b ligand ( rankl ) , and osteoprotegerin ( opg ) . through cell - to - cell contact of osteoblast / stromal cells with osteoclasts , rankl and m - csf induce osteoclast progenitor cells to differentiate into osteoclasts3 . bone morphogenetic protein ( bmp ) , a subgroup of the transforming growth factor- ( tgf- ) superfamily4 , induces the formation of bone and cartilage5,6,7,8,9,10 . to date , there are more than 20 known bmp subgroups , of which bmp-2 is the primary subgroup used as a clinical treatment in many in vitro and in vivo studies11 . recombinant human bmp-2 ( rh - bmp-2 ) is an activation factor for bone healing and has been used in dentistry due to its osteogenic effects . the aim of the present study was to explore the effect of rhbmp-2 on the regulation of opg , rankl , and m - csf in osteoclastogenesis in human osteoblast treated with alendronate . the following reagents were commercially obtained : pro - long gold antifade reagent with dapi , from molecular probes ( eugene , or , usa ) ; dulbecco 's modified eagle 's medium f12 ( dmem / f12 ) and fetal bovine serum ( fbs ) , from gibco ( gaithersburg , md , usa ) ; alendronate , dimethyl sulfoxide ( dmso ) , hoechst 33342 , rnase a , proteinase k , aprotinin , leupeptin , triton x-100 , pmsf and thiazolyl blue tetrazolium bromide ( mtt ) , from sigma ( st . louis , mo , usa ) ; supersignal west femto enhanced - chemiluminescence western blotting detection reagent , from pierce ( rockford , il , usa ) . an hfob 1.19 human fetal osteoblast cell line was purchased from the american type culture collection ( rockville , md , usa ) . the cells were maintained at 34 with 5% co2 in dmem / f12 with 4 mm l - glutamine , 1.5 g / l sodium bicarbonate , 4.5 g / l glucose and 1.0 mm sodium pyruvate supplemented with 10% fbs , under an air atmosphere . a total of 110 cells were seeded in a 96-well plate , incubated for 24 hours , and treated with 0 , 100 , 500 or 1,000 m concentrations of alendronate . the cells were then treated with 500 g / ml of mtt stock solution and incubated at 34 in a 5% co2 atmosphere for 4 hours . cell viability was monitored on an enzyme - linked immunosorbent assay ( elisa ) reader ( tecan , mnnedorf , switzerland ) at a 570 nm excitatory emission wave length . in cells treated with 100 m alendronate , expression levels of the three proteins , rankl , opg and m - csf , were measured with an elisa kit ( quantikine ; r&d systems , minneapolis , mn , usa ) according to the manufacturer 's instructions . briefly , cultured hfob 1.19 cells were uniformly seeded into 6-well culture dishes at a concentration of 210 cells / well . when the cells were adherent 24 hours later , the original medium was replaced with medium containing 100 m alendronate for 48 hours ( alendronate group ) after which 100 ng / ml bmp-2 ( cowellmedi , busan , korea ) was added , and the cells were incubated for a further 48 hours ( alendronate+rhbmp-2 group ) . the supernatants were then collected from each group , and an elisa was used to determine the rankl , opg and m - csf contents of each sample . the sample values were read from the standard curve set at 450 nm with 540 - 570 nm wavelength correction and with the measurement units expressed as pg / ml . all samples were assayed simultaneously . the hfob 1.19 cells were subjected to rna extraction using spin columns ( rneasy ; qiagen , hilden , germany ) according to the manufacturer 's instructions . rna ( 2 g ) was reverse - transcribed , using the revertaid first - strand synthesis system kit for rt - pcr ( thermo fisher scientific , pittsburgh , pa , usa ) according to the manufacturer 's protocol . the cdna was amplified with the pcr master mix sybr green kit ( applied biosystems , warrington , uk ) , and pcr amplification was performed using the chromo4 real - time pcr detection system ( bio - rad laboratories inc . , the running conditions were as follows : denaturing at 95 for 3 minutes , 40 cycles of amplification at 95 for 15 seconds and 60 for 30 seconds . after the final cycle , a melting curve analysis was performed at 55-95 intervals in 0.5 steps . the sense and anti - sense primer sequences of opg , rankl , m - csf , and the housekeeping gene glyceraldehyde 3-phophate dehydrogenase ( gapdh ) are listed in table 1 . tukey 's post hoc test was used for cell viability and rt - pcr ( spss statistics 17.0 ; spss inc . the following reagents were commercially obtained : pro - long gold antifade reagent with dapi , from molecular probes ( eugene , or , usa ) ; dulbecco 's modified eagle 's medium f12 ( dmem / f12 ) and fetal bovine serum ( fbs ) , from gibco ( gaithersburg , md , usa ) ; alendronate , dimethyl sulfoxide ( dmso ) , hoechst 33342 , rnase a , proteinase k , aprotinin , leupeptin , triton x-100 , pmsf and thiazolyl blue tetrazolium bromide ( mtt ) , from sigma ( st . louis , mo , usa ) ; supersignal west femto enhanced - chemiluminescence western blotting detection reagent , from pierce ( rockford , il , usa ) . an hfob 1.19 human fetal osteoblast cell line was purchased from the american type culture collection ( rockville , md , usa ) . the cells were maintained at 34 with 5% co2 in dmem / f12 with 4 mm l - glutamine , 1.5 g / l sodium bicarbonate , 4.5 g / l glucose and 1.0 mm sodium pyruvate supplemented with 10% fbs , under an air atmosphere . a total of 110 cells were seeded in a 96-well plate , incubated for 24 hours , and treated with 0 , 100 , 500 or 1,000 m concentrations of alendronate . the cells were then treated with 500 g / ml of mtt stock solution and incubated at 34 in a 5% co2 atmosphere for 4 hours . cell viability was monitored on an enzyme - linked immunosorbent assay ( elisa ) reader ( tecan , mnnedorf , switzerland ) at a 570 nm excitatory emission wave length . in cells treated with 100 m alendronate , expression levels of the three proteins , rankl , opg and m - csf , were measured with an elisa kit ( quantikine ; r&d systems , minneapolis , mn , usa ) according to the manufacturer 's instructions . briefly , cultured hfob 1.19 cells were uniformly seeded into 6-well culture dishes at a concentration of 210 cells / well . when the cells were adherent 24 hours later , the original medium was replaced with medium containing 100 m alendronate for 48 hours ( alendronate group ) after which 100 ng / ml bmp-2 ( cowellmedi , busan , korea ) was added , and the cells were incubated for a further 48 hours ( alendronate+rhbmp-2 group ) . the supernatants were then collected from each group , and an elisa was used to determine the rankl , opg and m - csf contents of each sample . the sample values were read from the standard curve set at 450 nm with 540 - 570 nm wavelength correction and with the measurement units expressed as pg / ml . all samples were assayed simultaneously . the hfob 1.19 cells were subjected to rna extraction using spin columns ( rneasy ; qiagen , hilden , germany ) according to the manufacturer 's instructions . rna ( 2 g ) was reverse - transcribed , using the revertaid first - strand synthesis system kit for rt - pcr ( thermo fisher scientific , pittsburgh , pa , usa ) according to the manufacturer 's protocol . the cdna was amplified with the pcr master mix sybr green kit ( applied biosystems , warrington , uk ) , and pcr amplification was performed using the chromo4 real - time pcr detection system ( bio - rad laboratories inc . the running conditions were as follows : denaturing at 95 for 3 minutes , 40 cycles of amplification at 95 for 15 seconds and 60 for 30 seconds . after the final cycle , a melting curve analysis was performed at 55-95 intervals in 0.5 steps . the sense and anti - sense primer sequences of opg , rankl , m - csf , and the housekeeping gene glyceraldehyde 3-phophate dehydrogenase ( gapdh ) are listed in table 1 . tukey 's post hoc test was used for cell viability and rt - pcr ( spss statistics 17.0 ; spss inc . , an mtt assay was performed at 48 hours with 0 , 100 , 500 , 1,000 m alendronate concentrations.(fig . when the cells were exposed to concentrations above 100 m , there was a significant decrease in cell viability ( p<0.05 ) . to investigate the reverse effects of rhbmp-2 on cell viability , cell viability assays were repeated with application of rhbmp-2 to cells treated with 100 m alendronate after 48 hours.(table 2 ) cell viability significantly increased relative to the control and alendronate alone group ( p<0.05 ) . to evaluate opg expression , rt - pcr and an elisa were performed . opg expression in the alendronate group was significantly decreased ( p=0.012 ) as compared to the control group . in the alendronate+rhbmp-2 group , the opg level increased relative to alendronate group , but this increase was not significant ( p=0.482).(fig . 2 , tables 3 , 4 ) rt - pcr and an elisa were also performed to evaluate rankl . in the alendronate group , rankl expression was significantly decreased ( p=0.003 ) as compared to the control . in the alendronate+rhbmp-2 group , rankl expression 3 , tables 3 , 4 ) to determine whether alendronate and rhbmp-2 treatment change the expression of m - csf , rt - pcr and an elisa were also done . in the alendronate group , m - csf expression maxilwas decreased significantly compared with the control group ( p=0.000 ) . in the alendronate+rhbmp-2 group , the m - csf level was decreased significantly compared with the control group but increased significantly compared with the alendronate group ( p=0.002).(fig . an mtt assay was performed at 48 hours with 0 , 100 , 500 , 1,000 m alendronate concentrations.(fig . when the cells were exposed to concentrations above 100 m , there was a significant decrease in cell viability ( p<0.05 ) . to investigate the reverse effects of rhbmp-2 on cell viability , cell viability assays were repeated with application of rhbmp-2 to cells treated with 100 m alendronate after 48 hours.(table 2 ) cell viability significantly increased relative to the control and alendronate alone group ( p<0.05 ) . opg expression in the alendronate group was significantly decreased ( p=0.012 ) as compared to the control group . in the alendronate+rhbmp-2 group , the opg level increased relative to alendronate group , but this increase was not significant ( p=0.482).(fig . rt - pcr and an elisa were also performed to evaluate rankl . in the alendronate group , rankl expression was significantly decreased ( p=0.003 ) as compared to the control . in the alendronate+rhbmp-2 group , rankl expression to determine whether alendronate and rhbmp-2 treatment change the expression of m - csf , rt - pcr and an elisa were also done . in the alendronate group , m - csf expression maxilwas decreased significantly compared with the control group ( p=0.000 ) . in the alendronate+rhbmp-2 group , the m - csf level was decreased significantly compared with the control group but increased significantly compared with the alendronate group ( p=0.002).(fig . we investigated the effects of rhbmp-2 on osteoblasts treated with alendronate , since alendronate is the most widely prescribed oral bp and appears most likely to cause bronj12 . the effects of bps on osteoclasts are well understood , and the effects of bps on osteoclastic toxicity are thought to influence the occurrence of bronj . although the majority of in vitro bps studies have focused on bps actions in osteoclastic lineage cells , recent studies have suggested that the presence of osteoblastic lineage cells is required for the anti - resorptive effects of bps13 . an understanding of the effects of alendronate on hfob cells , particularly in the opg / rankl system , however , is lacking . to address this lack of data , the current study investigated the expression of rankl , opg and m - csf in bp - treated hfob cells . here , alendronate was shown to have a strong negative dose - dependent influence on the viability of osteoblasts . mtt assays performed at 48 hours with 0 , 100 , 500 , and 1,000 m alendronate concentrations revealed that the 100 m concentration significantly reduced cell viability , with cell viability being approximately 80% of the control group . garca - moreno et al.14 reported that high concentrations of alendronate inhibit osteoblast proliferation in primary hfob cells and indicated that at lower concentrations the drug did not significantly inhibit proliferative effects compared to controls ( 10 m ) . the results of the current study are in agreement with garca - moreno et al.14 and further suggest that alendronate at higher concentrations affects proliferation and viability of hfob cells . we determined that expression of opg , rankl and mcsf was significantly decreased in the alendronate - treated group . in contrast , in a human osteoblast - culture study , koch et al.15 reported that zoledronate and ibandronate significantly induced rankl expression . sudhoff et al.16 reported that osteoclasts were suppressed and osteoclast apoptosis was induced in mouse bone - marrow cell ( bmc ) culture by the addition of m - csf , rankl and zolendronic acid . kwak et al.17 , having investigated a bmc / osteoblast co - culture , reported that risedronate inhibits osteoclast differentiation and suppresses rankl and that addition of m - csf mediates osteoclast differentiation in a bone - marrow - derived macrophage culture , with no direct bps - cytoxicity effect on the osteoclasts . although the reasons for these differences have not been completely elucidated , the distinct effects of various bps ( pamidronate , zoledronate , and alendronate ) , use at different concentrations , and use of different cell lines ( human vs. rat and primary vs. cancer ) clearly play a role15,18 bmp-2 is well recognized as the most effective boneinduction treatment19,20,21,22,23 and has been approved by the u.s . food and drug administration . itoh et al.27 reported that bmp-2 enhanced the survival of purified osteoclasts supported by rankl but not by m - csf . in our present investigation , we found that a lower dose of rhbmp-2 , 100 ng / ml , was able to induce osteoclast potency , indicating that at lower concentrations , bmp-2 can have osteoinductive potency28 . the alendronate+rhbmp-2 group showed significant elevation in expression levels of rankl and m - csf relative to the alendronate alone group . our results show that bmp-2 induced expression of m - csf but that rankl was otherwise suppressed by alendronate . this suggest that decreased rankl expression by bps can be corrected by treatment of bmp-2 . this study was conducted to establish whether alendronate and rhbmp-2 influence the mrna levels and protein expression levels of m - csf , rankl , and opg in osteoblasts . our results indicate that alendronate suppressed the expression of m - csf , rankl , and opg and that rhbmp-2 increased bp - impaired rankl , m - csf , and opg expression levels . together with the previous results , the present study provides an experimental basis for the clinical effects of rhbmp-2 as a bronj - treatment modality .

objectivesbisphosphonate - related osteonecrosis of the jaw ( bronj ) is a side effect of bisphophonate therapy that has been reported in recent years . osteoclastic inactivity by bisphosphonate is the known cause of bronj . bone morphogenetic protein-2 ( bmp-2 ) plays an important role in the development of bone . recombinant human bmp-2 ( rhbmp-2 ) is potentially useful as an activation factor for bone repair . we hypothesized that rhbmp-2 would enhance the osteoclast - osteoblast interaction related to bone remodeling.materials and methodshuman fetal osteoblast cells ( hfob 1.19 ) were treated with 100 m alendronate , and 100 ng / ml rhbmp-2 was added . cells were incubated for a further 48 hours , and cell viability was measured using an mtt assay . expression of the three cytokines from osteoblasts , receptor activator of nuclear factor-b ligand ( rankl ) , osteoprotegerin ( opg ) , and macrophage colony - stimulating factor ( m - csf ) , were analyzed by real - time polymerase chain reaction and enzyme - linked immunosorbent assay.resultscell viability was decreased to 82.75%1.00% by alendronate and then increased to 110.43%1.35% after treatment with rhbmp-2 ( p<0.05 , respectively ) . opg , rankl , and m - csf expression were all decreased by alendronate treatment . rankl and m - csf expression were increased , but opg was not significantly affected by rhbmp-2.conclusionrhbmp2 does not affect opg gene expression in hfob , but it may increase rankl and m - csf gene expression .

I. Introduction II. Materials and Methods 1. Reagents 2. Cell culture 3. Cell viability assay 4. ELISA 5. Real-time polymerase chain reaction (RT-PCR) 6. Statistical analysis Results 1. Viability of hFOB 1.19 cells 2. Effects of alendronate and rhBMP-2 on OPG expression 3. Effects of alendronate and rhBMP-2 on RANKL expression 4. Effects of alendronate and rhBMP-2 on M-CSF experssion Discussion Conclusion

PMC3820182

approximately 91% of anterior cruciate ligament ( acl ) injuries occur during sports activities1,2,3,4 ; most are caused by noncontact injury mechanisms , e.g. , landing from a jump and rapid deceleration5,6,7,8 . the position of the knee in noncontact acl injury is characterized by slight flexion ( < 30 ) , valgus of the knee , and internal rotation of the tibia1 , 9,10,11 . thus , training programs to prevent noncontact acl injuries should consider the anatomical and neurophysiological factors for preventing slight flexion and valgus of the knee joint . recent research has reported an association between the position of acl injuries and hip joint kinematics12 . hip and knee flexion angles upon landing are important factors determining the load on the knee , with slight flexion angles resulting in a greater load13 . moreover , the decrease in hip flexion angle during rapid deceleration results in a leg position that increases the quadriceps force , leading to anterior tibial displacement and increased risk of acl injury14 . these findings indicate that the shape of the femur may affect both kinematics of the hip and knee joints upon landing as well as muscle activity for protecting the joints . anteversion and the neck shaft angle are morphological factors of the femur that affect the kinematics of the hip and knee joints upon landing . femoral anteversion is the angle formed by the axis of the femoral neck and the horizontal axis of the femoral condyles . the angle of torsion decreases up to approximately 6 years of age , and the femoral head changes so that it faces more medially in the acetabulum15 . thus , when femoral anteversion is larger , the femoral head faces anteriorly in the acetabulum , resulting in reduced congruity of the hip joint . improved congruity of the hip joint may occur with excessive internal rotation of the hip16 and valgus of the knee17 . however , the association between neuromuscular control and hip and knee joint kinematics upon single - leg landing due to differences in femoral anteversion remains unclear . this study aimed to clarify the relationship between femoral anteversion and hip and knee joint kinematics and muscle activity of the lower extremity upon single - leg landing in terms of risk factors for acl injury . we hypothesized that increased femoral anteversion leads to an acl injury - risk position and , therefore , must be considered a risk factor for acl injury . sixteen healthy female college students ( age , 20.8 1.0 years ; height , 160.9 3.8 cm ; weight , 54.1 5.8 kg ) participated in this study . the purpose of the study and the measurements involved were explained to the subjects beforehand , both verbally and in writing , and their consent was obtained . this study was approved by the ethics committee of juntendo university graduate school of health and sports science . subjects performed a single - leg landing task by standing on both legs on a 30-cm high platform . they then jumped 30 cm forward , landing on the left leg . to minimize the effects of efforts for maintaining balance , subjects were instructed to cross their arms on their chest . inability to maintain this posture upon landing and allowing the opposite leg to contact the ground were considered failures . femoral anteversion was measured using craig 's test . for measurement , the knee joint of the tested leg the examiner palpated the greater trochanter while passively rotating the hip until the most prominent part of the greater trochanter reached its most lateral position . the angle between the shaft of the tibia and a line perpendicular to the floor the angle of the hip and knee joints upon single - leg landing was measured using the vicon mx three - dimensional ( 3d ) motion analysis system ( vicon motion systems , oxford , uk ) , eight infrared cameras , and a frequency of 100 hz . measurements were performed after the measurement error was confirmed to be 0.7 mm or less . ground reaction force was calculated synchronously at 1500 hz using an amti or6 - 7 force platform ( amti , watertown , ma , usa ) . infrared reflective markers were affixed to the body according to the marker positions in the plug - in gait lower body model ( vicon motion systems ) . markers were placed at 16 sites : both the anterior superior and posterior superior iliac spines , the center of both thighs externally , the lateral joint line of both knees , the center of both shanks externally , the lateral malleolus of both ankles , the center of both heels , and the head of the second metatarsal of both feet . marker trajectories were filtered with a woltring low - pass filter and a 20-hz cut - off frequency . ground reaction force data were filtered with a fourth - order butterworth low - pass filter with zero lag and a 6-hz cut - off frequency . the hip and knee joint angles in the sagittal and frontal planes were calculated using a plug - in biomechanical modeler ( vicon motion systems ) . the angle of hip flexion and extension were defined as the angles formed by the pelvic axis and femoral axis in the sagittal plane , with flexion denoted as + and extension denoted as . the angle of hip adduction and abduction were defined as the angles formed by the pelvic axis and femoral axis in the frontal plane , with adduction denoted as + and abduction denoted as . the angle of knee flexion and extension were defined as the angles formed by the femoral axis and shank axis in the sagittal plane , with flexion denoted as + and extension denoted as . the varus and valgus angles of the knee were defined as the angles formed by the femoral axis and shank axis in the frontal plane , with varus denoted as + and valgus denoted as . the measurement interval was calculated from the initial ground contact upon landing to 100 ms immediately after ground contact . initial ground contact was defined as the point at which the vertical ground reaction force exceeded 8 n. muscle activity of the lower extremity was measured using a telemyo 2400 surface electromyography system ( noraxon , scottsdale , az , usa ) and a 1500-hz sampling frequency . the electromyography ( emg ) data were collected from four muscles : the rectus femoris ( rf ) , gluteus maximus ( gm ) , semitendinosus ( st ) , and biceps femoris ( bf ) . the skin over the belly of each muscle was prepared for electrode placement by dry shaving and cleaning the area with alcohol to reduce surface impedance . blue sensor m-00-s surface electrodes ( ambu , ballerup , denmark ) affixed in the direction of the muscle fibers with a 2-cm interelectrode distance . the position of the electrode for rf was the midpoint of a line connecting the anterior superior iliac spine and the top of the patella , that for the gm was the midpoint of a line connecting the sacrum and the greater trochanter of the femur , that for the st was the midpoint of a line connecting the ischial tuberosity and the medial epicondyle of the tibia , and that for the bf was the midpoint of a line connecting the ischial tuberosity and the lateral epicondyle of the tibia . the emg data for each muscle were obtained from 100 ms before ground contact to 100 ms immediately after ground contact . preactivity plays a key role in maintaining knee joint stability after the impact of landing because the feedback mechanism alone is not enough to provide knee joint stability21 , 22 . thus , the activity approximately 100 ms before ground contact is crucial23 , 24 . acl strain upon landing reportedly peaks at approximately 40 ms after landing , and the ground reaction force after landing reportedly peaks at approximately 100 ms after landing4 , 25 , 26 . thus , muscle activity 100 ms after landing was measured because acl injury most likely occurs within 100 ms of landing . measurements of the maximum voluntary contraction ( mvc ) utilized the test positions for manual muscle testing reported by daniels and worthingham . normalization ( % mvc ) of the emg data for each muscle was accomplished using the emg data for 1 s during the waveform of mvc for 5 s. all measurements were performed for the left leg , and the mean of three attempts was used in the analysis . the mean for the 16 subjects was calculated on the basis of measurements of femoral anteversion ; the subjects were divided into two groups : the high group with six subjects at the upper end of the mean and the low group with six subjects at the lower end of the mean . although the femoral anteversions of the two groups were significantly different , there were no significant differences between the groups for age , height , and weight ( table 1 ) . all statistical tests were performed using the spss 11.0 j for windows statistical software ( spss inc . , the low and high groups were compared using unpaired t - tests for the kinematics and emg data . the alpha level for determining statistical significance gm , gluteus maximus ; rf , rectus femoris ; st , semitendinosus ; bf , biceps femoris mean sd . * p<0.05 . gm , gluteus maximus ; rf , rectus femoris ; st , semitendinosus ; bf , biceps femoris table 2 shows the mean hip and knee joint angles in the sagittal and frontal planes upon single - leg landing . the hip joint flexion from initial ground contact upon landing to 100 ms immediately after ground contact was significantly lower in the high group than in the low group ( p < 0.05 ; table 2 ) . significant differences between the two groups for hip joint adduction and abduction were not observed ( p < 0.05 ; table 2 ) . the knee joint flexion from 80 to 100 ms after initial ground contact was significantly higher in the high group than in the low group ( p < 0.05 ; table 2 ) . the knee valgus from 70 to 100 ms after the initial ground contact was significantly higher in the high group than in the low group ( p < 0.05 ; table 2 ) . muscle activity 100 ms before ground contact indicated that the % mvc of the gm was significantly less in the high group ( 0.5% 0.5% ) than in the low group ( 1.5% 0.6% , p < 0.05 ; table 3 ) . the % mvc of the rf was significantly greater in the high group ( 1.7% 0.5% ) than in the low group ( 0.9% 0.4% , p < 0.05 ; table 3 ) . significant differences between the groups in % mvc of the st and bf were not observed ( table 3 ) . muscle activity 100 ms after ground contact indicated that the % mvc of the rf was significantly greater in the high group ( 5.8% 0.7% ) than in the low group ( 4.0% 1.4% , p < 0.05 ; table 4 ) . significant differences between the two groups in % mvc of gm , st , and bf were not observed ( table 4 ) . abnormal alignment of the lower extremity has been proposed as a risk factor for acute and chronic lower extremity injuries such as acl injuries , patellofemoral syndrome , and plantar fasciitis . in addition , it has been suggested that biomedical changes caused by abnormal alignment of the lower extremity may influence joint load and muscles as well as neuromuscular control and function27 . increased femoral anteversion causes anterior displacement of the femoral head in the acetabulum and a decrease in congruity of the hip joint . thus , improvement in the congruity of the hip joint may occur with excessive internal rotation of the hip16 . excessive internal rotation of the hip leads to knee valgus alignment as a result of a kinematic chain17 ; thus , an increase in femoral anteversion may cause acl injury . our study results showed that the valgus of the knee in the high group increased after single - leg landing . this was because the congruity of the acetabulum and the head of the femur may have improved to protect the hip joint from the impact of landing ; excessive internal rotation of the hip may result in a kinematic chain . this study focused on joint kinematics in the frontal plane as well as the kinematics of the hip and knee joints in the sagittal plane . the hip and knee joint kinematics in the high group were characterized by lower hip joint flexion and higher knee joint flexion after single - leg landing . on the basis of video image analysis of acl injuries , a recent study reported that acl injury occurs when the trunk is positioned behind the leg28 . in other words , body position in the sagittal plane affects kinetic and kinematic components of the leg , presenting a risk of acl injury . blackburn and padua demonstrated that increased trunk extension upon landing increased the ground reaction force and the quadriceps forces29 . similarly , kulas et al . reported that increased trunk extension upon landing caused an increase in quadriceps forces and a decrease in hamstring forces30 . increased quadriceps forces lead to anterior tibial displacement , increasing the load on the acl28 , 31 . increased trunk extension is associated with a decrease in the angle of hip joint flexion32 . thus , increase of the femoral anteversion suggests a physical characteristic that probably leads to acl injury . the leg position upon landing is maintained by soft tissues such as muscles , the joint capsule , and ligaments . however , the feedback mechanism alone is not enough to provide knee joint stability and prevent injury21 , 22 . once the foot contacts the ground , the ground reaction force and acl strain peak . cerulli et al . reported that acl strain peaks approximately 40 ms after ground contact25 , whereas schmitz et al . reported that it increased approximately 100 ms after ground contact4 thus , acl injury occurs 40100 ms after ground contact , the time during which acl strain peaks . however , the feedback mechanism can not provide joint stability at this point ; therefore , the feedforward mechanism is important for joint stability23 , 24 . the results concerning emg activity in this study indicated lower muscle activity of the gluteus maximus and greater muscle activity of the rectus femoris before ground contact in the high group than in the low group . thus , the decrease in gluteus maximus activity before ground contact indicates that soft tissues such as ligaments are involved in limiting excessive hip internal rotation and that the ability to resist valgus of the knee diminished . the rectus femoris resists knee flexion moment32 and produces anterior tibial displacement32 , 35 , 36 . moreover , this muscle leads to valgus of the knee and internal rotation of the tibia37 ; thus , the rectus femoris increases the load on the acl . therefore , increased rectus femoris activity before and after ground contact may cause knee extensor moment and anterior tibial displacement . therefore , activity in these muscles may promote valgus of the knee and anterior tibial displacement , both of which predispose to acl injury . based on hip and knee joint kinematics upon single - leg landing and muscle activities immediately before and after ground contact , the above findings suggest that increased femoral anteversion may be a physical characteristic that probably leads to acl injury . furthermore , femoral anteversion may help to identify individuals who are likely to develop acl injury and may be considered a risk factor for acl injury . the kinematics of hip and knee joints upon single - leg landing were analyzed using the plug - in gait model . measurement of femoral anteversion is reportedly more reliable than techniques using x - rays38 . however , studies using more accurate measurement techniques involving sectional computed tomography images are required . in addition , kadaba et al . indicated that disparities in the placement of infrared reflective markers in the plug - in gait model can lead to errors ; i.e. , errors in measurement of joint motion other than knee joint flexion / extension39 . future studies should validate the knee joint motion in detail using the point cluster technique40 , which allows more accurate measurement of the 3d motion of the knee .

[ purpose ] increased femoral anteversion may occur with hip internal rotation and valgus knee alignment upon landing and is considered a risk factor for anterior cruciate ligament injury . we examined the relationship between femoral anteversion and joint motion and muscle activity of the lower extremity in terms of the risk factors for anterior cruciate ligament injury . [ subjects ] sixteen healthy females were divided on the basis of femoral anteversion into low and high groups . [ methods ] femoral anteversion was assessed using craig 's test . we performed kinematic analysis and measured the electromyography activity of the lower extremity upon left single - leg landing . [ results ] the high group had a significantly lower hip flexion angle and higher knee flexion and valgus angles than the low group . the rectus femoris showed significantly greater electromyography activities in the high group than in the low group . [ conclusion ] these results suggest that increased femoral anteversion results in lower hip flexion angle , higher knee valgus alignment , and greater rectus femoris muscle activity , leading to anterior tibial displacement upon single - leg landing . increased femoral anteversion may be a potential risk factor for anterior cruciate ligament injury .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3560548

small intestinal mucosa , which forms the intestinal pouch , undergoes several adaptive processes aimed at taking over some of the functions of the removed large intestine . apart from adaptive changes , a significant number of patients may experience a certain type of inflammation . several potential etiopathological causes of pouchitis might be listed , including static fecal residue in the pouch in patients and experimental models . this phenomenon has also been explained by the overgrowth of bacteria , but the latest research does not confirm this theory . it has been observed that endoscopic dilatation of the stricture decreases the clinical symptoms and improves comfort . whether the anastomotic stricture is the cause of pouchitis or the result of an inflammatory condition which induces fibrosis is an issue that still raises many doubts and all surgical procedures were conducted by a qualified surgeon in accordance with the guidelines of the european community council directives 86/609/eec and with the agreements of the local ethics committee ( 42/2006 ) . adult male wistar rats in good general condition ( 300350 gms ) were housed individually in stainless steel metabolic cages under controlled temperature ( 2123c ) on 12 : 12-h light - dark cycle and fed with standard laboratory diet and water ad libitum . in all examined animals , feeding was stopped 24 hours before surgery . after an intraperitoneal injection of pentobarbital anesthesia ( pentobarbital sodium , 50 mg / kg body wt ) the skin of the abdomen was sterilized and draped with gauze and a 34 cm midline laparotomy was performed . the total proctocolectomy was performed by resecting the colon and ligating the mesentery with 4 - 0 silk . the rectum was resected at the level of the pelvic floor , with a 0.5 cm rectal stump . small intestinal contents were washed with 0.9% warm saline and a 2 cm ileal j - pouch was created by the duplication of the distal end of the small intestine by a single - layer interrupted 6 - 0 prolene suture . the pouch anal anastomosis was performed with a single layer interrupted 6 - 0 prolene suture . the sufficiency of the anastomosis was controlled by injection of 35 ml of 0.9% warm saline through the anus . i ( 11 cases ) normal width anastomosis was performed , assuming 75% of the diameter of the distal part of small intestine to be ( 12 cases ) the diameter of anastomosis was reduced by 50% in comparison to group i ( figure 1 ) . the abdominal incision was closed with a running 2 - 0 silk suture and the skin with a single - layer interrupted 3 - 0 silk suture . after 9 weeks of postoperative follow - up , all animals were sacrificed by decapitation . the mucosa specimens with an area of 0.5 cm were fixed in formalin for further histological and immunohistochemical evaluations . each time the histopathological examination was performed by 2 independent histopathologists and every time the examined section was found to be inflamed according to moskowitz scale and positive for villi atrophy according to laumonier scale . histological classification of inflammation suggested by moskowitz objectively presents the microscopic assessment of acute and prolonged inflammation and ascribes certain points , which we sum up , to particular features . based on hematoxylin & eosin preparations , paraffin blocks containing sections with the most characteristic changes were selected for further immunohistochemical tests . to demonstrate the expression of the investigated markers ( il-1 alpha , il-6 , il-10 , il-12 ) , the streptavidin - biotin - peroxidase method was employed using an lsab kit manufactured by dako . the antigen was located using dab-3.3 ( dako ) as a chromogen , which is a substrate for peroxidase . the preparations were then stained with hematoxylin and following dehydration closed with a glass cover . the differences in the anastomosis diameter , intensity of pouchitis and interleukin expression between the 2 selected groups of animals were calculated with the use of the mann - whitney u test . adult male wistar rats in good general condition ( 300350 gms ) were housed individually in stainless steel metabolic cages under controlled temperature ( 2123c ) on 12 : 12-h light - dark cycle and fed with standard laboratory diet and water ad libitum . in all examined animals , feeding was stopped 24 hours before surgery . after an intraperitoneal injection of pentobarbital anesthesia ( pentobarbital sodium , 50 mg / kg body wt ) the skin of the abdomen was sterilized and draped with gauze and a 34 cm midline laparotomy was performed . the total proctocolectomy was performed by resecting the colon and ligating the mesentery with 4 - 0 silk . the rectum was resected at the level of the pelvic floor , with a 0.5 cm rectal stump . small intestinal contents were washed with 0.9% warm saline and a 2 cm ileal j - pouch was created by the duplication of the distal end of the small intestine by a single - layer interrupted 6 - 0 prolene suture . the pouch anal anastomosis was performed with a single layer interrupted 6 - 0 prolene suture . the sufficiency of the anastomosis was controlled by injection of 35 ml of 0.9% warm saline through the anus . i ( 11 cases ) normal width anastomosis was performed , assuming 75% of the diameter of the distal part of small intestine to be ( 12 cases ) the diameter of anastomosis was reduced by 50% in comparison to group i ( figure 1 ) . the abdominal incision was closed with a running 2 - 0 silk suture and the skin with a single - layer interrupted 3 - 0 silk suture . after 9 weeks of postoperative follow - up , all animals were sacrificed by decapitation . the mucosa specimens with an area of 0.5 cm were fixed in formalin for further histological and immunohistochemical evaluations . each time the histopathological examination was performed by 2 independent histopathologists and every time the examined section was found to be inflamed according to moskowitz scale and positive for villi atrophy according to laumonier scale . histological classification of inflammation suggested by moskowitz objectively presents the microscopic assessment of acute and prolonged inflammation and ascribes certain points , which we sum up , to particular features . based on hematoxylin & eosin preparations , paraffin blocks containing sections with the most characteristic changes were selected for further immunohistochemical tests . to demonstrate the expression of the investigated markers ( il-1 alpha , il-6 , il-10 , il-12 ) , the streptavidin - biotin - peroxidase method was employed using an lsab kit manufactured by dako . the antigen was located using dab-3.3 ( dako ) as a chromogen , which is a substrate for peroxidase . the preparations were then stained with hematoxylin and following dehydration closed with a glass cover . the differences in the anastomosis diameter , intensity of pouchitis and interleukin expression between the 2 selected groups of animals were calculated with the use of the mann - whitney u test . higher postoperative mortality ( 5 cases ) was found in group ii as compared with group i ( 2 cases ) , but the difference did not reach the level of significance . the mean diameter of anastomosis in group i was 4.2 mm and in group ii was 2.3 mm ; the differences were statistically significant ( p=0.0086 ) . morphological assessment of pouchitis symptoms based on moskowitz scale revealed considerably more severe inflammation ( p=0.0079 ) in the rats from group ii than in the rats from group i. no significant differences related to intestinal villi atrophy were observed . the expressions of investigated cytokines , assessed qualitatively in histopathological examination , were higher in rats with narrow anastomosis in comparison with animals with normal anastomosis . the differences in il-10 were statistically significant ( p=0.0115 ) and differences in il-1 , il-6 , il-12 showed no statistical significance . clinical research on pouchitis is limited by the number of patients , their biological and clinical differences , and their willingness to participate in the follow - up examinations , which are often invasive and have to be performed within particular time periods . thus , appropriate experimental models are needed in order to enable the better understanding of mechanisms of pouchitis and , consequently , its more effective treatment . it is also believed that pouchitis is a natural model of ibd , with a defined onset time within mucosa . pig models have long been used in research on restorative proctocolectomy for the assessment of inflammatory manifestations , bacteria colonization or post - operative peristalsis in the digestive tract . these models allow for the reconstruction of surgical procedures in humans but they are relatively rarely used due to their sizes and related costs , as well as other restrictions , . rat models are used much more frequently , in which either idiopathic pouchitis is assessed or it is chemically induced , most frequently with the use of 5% dextran sulfate sodium ( dss ) , iodoacetamide or dinitrobenzene sulfonic acid ( dnbs ) . the rat experimental models of pouchitis pose a vast array of surgical difficulties related to the necessity of the performance of a low anastomosis . some trials aimed at technical modifications have already been described , which allow for simplification of surgical procedures and the avoidance of a low anastomosis . one of them is to create a u - shaped ileal pouch ( analogous to a classical proctocolectomy ) and anastomose it to the intestine anterior to the ileocecal valve ( without colectomy being performed ) . clinical observations reveal that anastomotic stricture is one of the most significant problems of restorative proctocolectomy patients , and its endoscopic dilatation decreases the clinical symptoms as well as improving comfort . presently , more accurate data is available that allows for more precise definition of anastomotic stricture . it is now assumed that an 8 mm diameter is a critical anastomotic stricture after restorative proctocolectomy . there is still a lack of data about the correlations between the diameter of the anastomosis and the occurrence and the severity of pouchitis . therefore , the model of anastomotic stricture , which is described in our research , was regarded as provisional and based on the team s previous experiences . the starting point was a standard performance of anastomosis , whose diameter corresponded to 75% of the diameter of the distal part of the small intestine in the part slightly above the pouch . in analogy to clinical data , the stricture was assumed to have an anastomotic diameter equal to 50% or less of the normal diameter . on the basis of our research it has been concluded that the group of patients with narrow anastomosis presented more severe pouchitis assessed in accordance to the moskowitz histological classification . anastomotic stricture leads to static fecal residue , which is one of the potential causes of pouchitis in clinical and experimental models . in the examined sections , no divergences related to the atrophy of intestinal villi , in accordance with laumonier classification , atrophy of intestinal villi is a long - term process indicating the continuing restructuring of the mucosa of the small intestinal reservoir . in restorative proctocolectomy patients thus , in our research the time for observation might have been insufficient to note the increased atrophy of intestinal villi and potential differences among the groups . in the present study we also evaluated the expression of several interleukins in the mucosa of the intestinal reservoir . interleukins known for their pro - inflammatory properties , such as il-1 , il-6 and il-12 , as well as anti - inflammatory il-10 , were assessed . however , in cases of pro - inflammatory cytokines ( il-1 , il-6 , il-12 ) , these differences were statistically insignificant . obviously , the procedure itself was the factor that greatly influenced the activity of assessed interleukins . the phenomenon of increased activity of pro - inflammatory ( il-6 ) and anti - inflammatory ( il-10 ) interleukins was observed in peritoneal cavity exudates in patients with anastomotic leakage after partial resection of the large intestine . the increased level of il-6 and il-10 was noted even in cases of non - complicated large intestine partial resection . the increased level of il-10 is of particular interest . despite its known anti - inflammatory properties , the role of this interleukin in the development of pouchitis has not been thoroughly examined . there is some evidence supporting its anti - inflammatory activity in pouchitis , yet it has also been noted that its level was similar in patients with pouchitis and healthy subjects . it seems that significantly higher levels of il-10 activity in rats with narrow anastomosis may be related to higher inflammatory reaction , which is a non - specific stimulus to il-10 and , to a smaller extent , to pro - inflammatory interleukins . narrow anastomosis significantly influences the intensity of inflammatory changes within the pouch mucosa and stimulates the production of interleukins , in particular the anti - inflammatory il-10 . obtained results suggest that recommendations in pouchitis should take into consideration the assessment of the anastomosis width and its dilatation in justified cases .

summarybackgroundpouchitis appears to be the most common complication after restorative proctocolectomy.material/methodsin experimental models we investigated the correlation between the width of anastomosis and the frequency of pouchitis . twenty - three wistar rats underwent restorative proctocolectomy under pentobarbital anesthesia . normal width anastomosis was performed in 11 animals ( group i ) . in the remaining 12 animals ( group ii ) the diameter of anastomosis was reduced by 50% . all animals were sacrificed and the pouch mucosa was histologically ( moskowitz score ) and immunohistochemically ( il-1 , il-6 , il-10 , il-12 expression ) examined.resultsmorphological assessment of pouchitis symptoms based on moskowitz scale revealed considerably more severe inflammation ( p=0.0079 ) in the animals from group ii than in the rats from group i. the expressions of investigated cytokines , assessed qualitatively in histopathological examination , were higher in rats with narrow anastomosis in comparison with animals with normal anastomosis.conclusionsthe stricture of anastomosis increases the intensity of pouchitis and stimulates the production of interleukins . it seems that anastomotic stricture plays an important role in the development of pouchitis .

Background Material and Methods Surgical treatment Collection of the specimens Histological assessment Immunohistochemistry Statistical analysis Results Discussion Conclusions

PMC4668130

a change of lifestyle entailing increased physical activity , quitting smoking and dietary modifications is an effective and inexpensive way of improving individuals health and lowering their cardiovascular risk1,2,3,4 . physical exercise can be prescribed as therapy in two forms : unsupervised and supervised5 . there are many models of rehabilitation , even within the scope of treating and preventing cardiovascular diseases1 , 4 , 6 , 7 . however , the most important provision is that daily physical activity should not be shorter than thirty minutes3 , 4 . one of the new and increasingly popular forms of exercise is nordic walking which is a combination of walking and cross - country skiing , walking assisted with poles8 . the most important elements of nordic walking exercise comprise : thrusting the walking pole , pushing oneself off with the arm holding the pole and lifting a leg off the ground . these movements , combined in a fluent technique , influence the speed of walking and its effectiveness . nordic walking utilizes 90% of all muscles ( including lower and upper extremity muscles ) , strengthens the muscles of the upper part of the body and shoulders and increases the mobility of the upper segment of the spine . walking poles relieve stress on the joints , straighten and relieve stress on the spine and correct faulty posture by strengthening the stabilizing muscles9 , 10 . when preforming nordic walking calorie consumption is 2030 or even 67% more intense than during running . so far 16 randomized ( 1,062 participants ) and 11 observational studies ( 831 ) of nordic walking have been carried out . these studies have confirmed the positive effects of nordic walking on resting cardiac activity , arterial blood pressure , exercise tolerance , exercise oxygen consumption and quality of life in the course of various diseases of young13 , middle - aged14 and elderly persons8 , 15,16,17 . it was confirmed that this model of rehabilitation , in particular the supervised one5 , extends the walking distance of patients suffering from atherosclerosis in the lower extremities18 , improves the fitness of patients suffering from parkinson s disease19 , 20 , reduces low back pain5 , 21 , symptoms of depression22 and fibromyalgia23 , 24 , improves the quality of sleep , reduces body weight , improves exercise tolerance and attenuates risk factors . however , these effects pertain primarily to persons with normoglycemia rather than overweight patients suffering from glucose intolerance or diabetes type 216 , 25,26,27,28 and a four - month nordic walking rehabilitation program did not improve the level of glycosylated haemoglobin ( hba1c ) in patients suffering from diabetes type 2 and obesity29 . menopause is connected with numerous hormonal changes which can lead to obesity , lipid and carbohydrate metabolism disorders as well as related increases in risk of cardiovascular events and death . increasing physical activity is one of the methods used to reduce these risks1 , 3 . the beneficial influence of 34 cardiological rehabilitation programs on the cardiovascular risk ( or= 0.64 , 95%ci= 0.460.88 ) and general mortality was recently confirmed in the meta - analysis carried out by lawler et al6 . the positive influence of exercise stems , among other things , from reduction in body weight and insulin resistance which , can result in improvement or even normalization of lipid and carbohydrate metabolism disorders2 . it was demonstrated that exercise generally lowers the concentration of triglycerides and increases the concentration of hdl cholesterol2 . however , as yet there is not much data on the hypolipidemic effect of nordic walking rehabilitation11 , 30 . this is an issue of great importance as hyperlipidemia and obesity are the main modifiable risk factors of atherosclerosis in poland . a study carried out by natpol plus showed that hyperlipidemia and obesityaffect 61 and 53% of persons , respectively31 , 32 . the aim of this study was to evaluate the effect of a ten - week nordic walking rehabilitation program on chosen anthropometric parameters and the level of basic lipids in overweight and obese postmenopausal women s blood . the study subjects were 32 women with an average age of 59.7 5.9 years ( age range : 5068 ) . their basic demographic and clinical data are presented in table 1table 1.measured values at the beginning of the study and after the ten - week nordic walking programparameterat the beginningof the studyafter 10 weeksbody weight ( kg)80.7 14.076.2 14.0 bmi ( kg / m)30.5 4.128.8 4.5 cpk ( u / l)109 47.090 37 total cholesterol ( mg / dl)213.3 34.5194.1 28.4 ldl cholesterol ( mg / dl)133.5 28.8117.2 30.2 hdl cholesterol ( mg / dl)59.4 13.064.9 13.3 triglycerides ( mg / dl)124.6 46.597.8 33.1 quotient of total cholesterol and hdl3.7 0.83.1 0.7 percentage of participants with a total cholesterol concentration of < 190 mg / dl ; n ( % ) 8 ( 25%)12 ( 37.5% ) percentage of participants with ldl cholesterol concentration of < 115 mg / dl ; n ( % ) 7 ( 22%)16 ( 50% ) percentage of participants with hdl cholesterol concentration of 46 mg / dl ; n ( % ) 28 ( 87.5%)32 ( 100% ) percentage of participants with a triglyceride concentration of < 150 mg / dl ; n ( % ) 23 ( 71%)29 ( 91% ) percentage of participants with a quotient of total cholesterol concentration and hdl < 5 ; n ( % ) 30 ( 94%)32 ( 100%)cut - off points for concentration of lipids were those of the european society of cardiology2 , 3 . statistically significant differences between the initial value and after the 10-week nordic walking program . * p<0.05 ; p<0.001 . none of the participants suffered from serious somatic or mental diseases . additionally , the participants in this study did not take any medication other than metabolically neutral hypotensive drugs . cut - off points for concentration of lipids were those of the european society of cardiology2 , 3 . statistically significant differences between the initial value and after the 10-week nordic walking program . the study was carried out following a non - randomized and unsupervised medical experiment model . the primary intervention consisted of a nordic walking rehabilitation program carried out under a trainer s supervision . session were conducted 5 times a week for 10 weeks ( in total : 50 sessions , 60 minutes each ) and they complied with the minimum exercise requirements recommended by the european society of cardiology ( esc ) . prior to commencing the program in the nordic walking training rehabilitation technique was carried out and participants were tested on their mastery of it . a warm - up was organized before each training session . at the beginning the average distance covered amounted to 3.8 km . as progress in rehabilitation was made and exercise tolerance improved , the distance increased to 5.3 km . the average , estimated energy expenditure during each session was 450 kcal . during the entire period of the study each woman was on the same diet ( meals consumed in catering establishments ) . the participants daily diet consisted of 5 meals with a caloric value of 1,500 kcal . energy sources were planned as follows : complex carbohydrates 50% , fat 30% , protein 20% . the participants did not change their medication as it could have that could influenced the studied parameters . all the participants received a medical examination at the beginning and at the end of the study . during the examination anthropometric parameters ( weight , height , body mass index ) the concentration of total cholesterol , ldl , hdl and triglycerides as well as creatine phosphokinase activity ( cpk , a skeletal muscle damage marker ) was examined before and after intervention . the measurement of biochemical parameters was performed using blood serum collected in the morning , before training , from the cubital fossa vein , with minimum tourniquet pressure , 14 hours after the last meal and after 15 minutes of rest . the effects of the rehabilitation program were measured through the changes in the values of the anthropometric and biochemical parameters described above . the absolute difference delta and relative changes in parameters ( percentage change ) were calculated . this study was performed with the approval of the bioethical commission of the nicolaus copernicus university in toru , ludwik rydygier collegium medicum in bydgoszcz ( no . kb/602/2011 ) in september 2011 . the results are presented as the mean value and standard deviation or the median and range as well as the number ( n ) or percentage of participants depending on the type of variable ( quantitative , qualitative ) and normality of their distribution . the significance of difference in the values of categorical variables and variation was carried out using the wilcoxon test . the mann - whitney u test was used to test differences between subgroups determined by the median age value ( < i 62 years ) , body weight ( < i 75.4 kg ) , height ( < i 162.5 cm ) and bmi ( < i 30 kg / m ) . additionally , the percentage of participants , who achieved the recommended levels of lipids recommended for female subject to a medium risk of cardiovascular diseases ( total cholesterol < 190 mg / dl , ldl < 115 mg / dl , hdl46 mg / dl , triglycerides < 150 mg / dl)2 , 3 was calculated . a comparison of the significance of differences within the scope of the size of groups was carried out using fisher s exact test . after 10 weeks of exercises performed according to the nordic walking model statistically significant reductions in body weight ( on average 4.5 kg , 5.7% ) and bmi ( on average by 1.7 kg / m , 5.7% ) were observed . eighteen participants ( 56% ) lost less than 5% of their body weight , 9 participants ( 28% ) lost 510% of their body weight and 5 ( 16% ) lost 10% of their body weight . in the biochemistry results statistically significant drops in total cholesterol ( median delta 19.2 mg / dl , 8.5% ) , ldl ( median delta 16 mg / dl ; 12% ) , and triglyceride concentrations ( median delta 26.8 mg / dl ; 0.8% ) were observed . additionally , the concentration of hdl cholesterol increased ( median delta 5.5 mg / dl , 10% ) ( table 1 ) . after the rehabilitation program had ended , the percentage of participants with the recommended levels of total cholesterol , ldl , hdl and triglycerides had significantly increased ( table 1 ) . the changes in lipid concentrations in blood serum observed after completion of the 10-week nordic walking program showed considerable individual variation and the ranges are presented in table 2table 2.the absolute ( delta ) and relative ( % ) changes in the outcome measures after a 10-week nordic walking programparametermean value standarddeviation95%ciof the mean valuemedianscope(minimum - maximum)d_weight ( kg)4.5 3.45.7 to 3.32.9511.0 to 0.5d_weight_% ( % ) 6 47.3 to 4.13.315 to 0.6d_bmi ( kg / m)1.7 1.22.1 to 1.21.154.2 to 0.2d_bmi_% ( % ) 6 47.3 to 4.13.315 to 0.7d_total cholesterol ( mg / dl)19.2 17.525.5 to 12.912.068.0 to 2.0d - total cholesterol_% ( % ) 8.5 711 to 66.028 to 0.09d - ldl ( mg / dl)16.3 17.722.7 to 9.911.066.0 to 5.0d_ldl_% ( % ) 12 1116.3 to 7.88.850 to 2.9d_hdl ( mg / dl)5.5 6.23.2 to 7.75.02.0 to 30.0hdl_% ( % ) 10 11.56.0 to 14.39.33.7 to 55.6d - triglycerides ( mg / dl)26.8 25.436.0 to 17.716.586.0 to 0.00d_triglycerides_% ( % ) 19.2 1524.5 to 14.015.655 to 0.00d_tc / hdl(u / i)0.61 0.410.76 to 0.460.522.0 to 0.03d_tc / hdl_% ( % ) 16 919.5 to 12.813.642 to 0.6delta ( d ) = difference at the end of the study presented at the absolute value ( d_parameter ) and the relative value ( parameter_% ) , the percentage change with respect to the initial value ; tc / hdl quotient of total cholesterol and hdl concentration ( the atherogenic lipids in blood serum index ) ; 95% ci confidence interval 95% .. this is the reason influence of chosen clinical and anthropometric factors on the values of studied parameters was evaluated before commencing and after complete on of the nordic walking program . the values of the studied biochemical parameters were compared in subgroups of women determined by on the median age ( < i 62 years ) , body weight ( < i 75.4 kg ) , height ( < i 162.5 cm ) and bmi ( < i 30 however , statistically significant differences were found only between the group of overweight and obese women . overweight participants ( bmi 30 kg / m ) experienced a considerably lower bmi reduction ( 1.3 vs. 2.1 kg / m ; 3.6 vs. 7.8% ; p=0.045 ) . the initial bmi value correlated with its percentage ( relative ) decrease after completing the intervention ( r = 0.39 , p = 0.026 ) . the absolute decrease in bmi , on the other hand , correlated with the initial height of participants . the participants , whose weight loss exceeded 5% showed bigger absolute ( 8.5 8.1 vs. 3.1 2.5 mg / dl ; p = 0.012 ) and relative ( 15.7 15.0% vs. 5.8 4.7% ; p = 0.012 ) increases in hdl concentration after 10 weeks of nordic walking rehabilitation . delta ( d ) = difference at the end of the study presented at the absolute value ( d_parameter ) and the relative value ( parameter_% ) , the percentage change with respect to the initial value ; tc / hdl quotient of total cholesterol and hdl concentration ( the atherogenic lipids in blood serum index ) ; 95% ci confidence interval 95% . our results show that after ten weeks of nordic walking rehabilitation 32 overweight and obese postmenopausal women showed a statistically significant loss of body weight , as well as a drops in bmi and concentrations of atherogenic lipids in blood serum . after completing the program the percentage of participants with the recommended levels of total cholesterol , ldl , hdl and triglycerides drastically increased ( table 1 ) . a statistically significant weight loss ( > 5% ) was observed among 44% of the participants , also showed an increase in hdl cholesterol concentration . moreover , the results demonstrate that participants with a higher degree of obesity experienced a smaller reduction in bmi . there was no statistical correlation between initial bmi and the hypolipidemic effect of the rehabilitation program . weight loss after 1012 weeks of nordic walking was also observed by another group of researchers30 . however , although our program was shorter ( 10 vs. 16 weeks ) , we observed body weight loss and bmi reduction26 . goodpaster et al.33 carried out a randomized single - blind trial with a one - year observation period , durning which they investigated whether moderately strenuous exercise , of an intensity similar to a quick march , performed 5 times a week for 60 minutes by morbidly obese subjects could increase weight loss compared to diet alone . after 6 months of controlled exercise and dietary intervention , it 80% of the participants showed a 5% weight loss to 60% of the participants who were only advised to change their diet . in our study this suggests that it is necessary to implement a dietary intervention in order to increase the effectiveness of weight loss . it might be worth involving more specialists ( for example a bariatrist , a bariatric surgery specialist , a psychologist , a psychiatrist , etc . ) who would cooperate just like a cardiological heart team . our study also showed that a greater weight loss was observed among taller participants which confirms the negative correlation between energy expenditure when doing nordic walking and the length of the poles previously reported in the literature12 . therefore , it would be beneficial to further analyse the usefulness of complex programs ( dietary intervention , exercises , behavioural therapy ) in treating obesity with respect to the reduction in cardiovascular risk , even when weight loss is small ( 510%)33 . as mentioned above , the 10-week nordic walking rehabilitation program had a positive effect on the level of basic blood lipids in overweight and obese postmenopausal women ( table 1 ) . the nordic walking program reduced the total cholesterol , ldl and triglyceride concentrations and increased the concentration of the anti - atherogenic fraction of hdl ( table 2 ) . after completion of the rehabilitation program , concentrations of total cholesterol and ldl were lower on average by 19 ( 9% ) and 16 ( 12% ) , respectively . however , previous epidemiological observations3 , 35 , 36 and the results of multicentre randomized intervention studies justify the hypothesis that the observed effects of lipid rehabilitation in the nordic walking model have noticeable health - related benefits . it is generally accepted that as far as the treatment of the obesity is concerned , health - related advantages are achieved even when weight loss is small 510%33 . moreover , it is known that an increase in total cholesterol of 1% leads to an increase in the cardiovascular event risk of 2%35 , 36 . a drop in ldl concentration of 1 mmol / l ( approx . 40 mg / dl ) during statin therapy , on the other hand , correlates with a 10% reduction in the total mortality and a 22% reduction in the risk of death and cardiovascular morbidity rate2 . in addition , whereas the probability of a serious coronary event reduces by 23% , the risk of cerebral stroke reduces by 17% . a further potential benefit of rehabilitation through nordic walking arises from the increase in hdl cholesterol concentration . an increase in hdl of 1 mg / dl is reported to reduce the cardiovascular risk by 23%37 . in the present study , the increase in hdl cholesterol concentration amounted on average to 5.5 mg / dl ( 10% ) which would correspond to an average reduction in cardiovascular death of 1015% . a higher average increase in hdl was achieved by participants whose weight loss exceeded 5% . these results are consistent with the data presented in the literature showing that weight loss of 1 kg is associated with an increase in the hdl cholesterol concentration of 0.4 mg / dl2 . an increase in hdl cholesterol can also be achieved through regular physical activity and quitting smoking2 . it is worth mentioning that the most popular hypolipidemic medicine , statins , increase the level of hdl in a similar way , i.e. by 510%2 . some authors are of the opinion that the level of triglycerides after a meal is a better determinant of cardiovascular risk38 . in the present study triglycerides were determined on an empty stomach , and their level after 10 weeks of practising nordic walking was 27 mg / dl ( 10% on average ) . according to the literature , reduction in body weight through physical activity should lower the level of triglycerides by 2030%2 . the fact that our results were poorer than this can be explained the lack of a dietary intervention . nevertheless , the overall clinical results of the proposed nordic walking rehabilitation program were associated with its hypolipidemic effect , and can be expressed as the potential reduction in cardiovascular risk , estimated to be in the range of least 2025% ( 10% dependent on ldl reduction and 1015% dependent on hdl increase ) , before considering the advantages of the workout and weight loss . the described effect is achieved with the help of an average dose of statin2 . yet , during the study the response to the suggested rehabilitation program varied significantly among individuals ( table 2 ) . consequently , future studies of the clinical effectiveness of nordic walking are warranted in order to identify subjects who apart from physical activity , require an additional intervention to reduce their cardiovascular risk . there is also no certainty that the meals consumed by participants were the only food they consumed . additionally , we did not use the risk score table to evaluate the initial and final cardiovascular risk . in conclusion , a ten - week nordic walking workout program resulted in a relatively small , yet statistically significant , weight loss , significant reductions in atherogenic serum lipids levels , and an increase in anti - antherogenic hdl cholesterol levels as well as a drop in the skeletal muscle damage index ( cpk ) . the percentage of participants with the recommended level of lipid fractions had increased at the end of the intervention . obese participants showed relatively lower weight loss at the end of the intervention than overweight subjects , and those who lost more than 5% of their body weight showed a bigger hdl cholesterol concentration increase and the end of the nordic walking program . overweight and shorter participants probably require a more intense workout and cooperation with a dietician to achieve a statistically significant weight loss .

[ purpose ] the aim of this study was to evaluate the effect of a ten - week nordic walking ( nw ) rehabilitation program on chosen anthropometric parameters and the level of basic lipids in overweight and obese postmenopausal women s blood . [ subjects and methods ] the subjects were 32 women aged 5068 ( average : 59.7 5.9 years ) . the study was carried out following a non - randomized model and entailed nw rehabilitation 5 times a week , which lasted for 10 weeks , as well as a low - calorie 1,500 kcal diet . the therapeutic results of the study were measured through changes in anthropometric and biochemical parameters . the results were subjected to a statistical analysis . [ results ] after 10 weeks of nw rehabilitation it was observed that participants lost weight and their body mass index dropped . additionally , whereas levels of total cholesterol , ldl and triglycerides dropped , and the level of hdl increased . [ conclusion ] rehabilitation carried out according to the nw model resulted in statistically significant changes in basic lipids in blood which , considerably increased the percentage of persons who achieved the recommended level of blood lipids . obese persons were characterised by a smaller rehabilitation weight loss . more intense workouts and cooperation with a dietician are required .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC2943102

the regulation of energy intake in humans is based on a series of complex mechanisms that is not solely driven by homeostatic factors such as hunger and satiety signals . it has been suggested that cognitions and emotions are largely involved in the regulation of energy intake . different tools have been proposed to grasp the complexity of eating behaviour traits in humans . among them , the three - factor eating questionnaire developed by stunkard and messick assesses three dimensions of eating behaviour traits : dietary restraint , disinhibition , and hunger . briefly , dietary restraint is defined as a conscious control of food intake with concerns about shape and weight , disinhibition refers to an overconsumption in response to a variety of stimuli associated with a loss of control on food intake , and hunger is the food intake in response to feelings and perception of hunger . this questionnaire has been widely used to study the association between eating behaviour traits and body weight . the association between dietary restraint and ( bmi ) is uncertain ; some authors have found no association [ 58 ] while others have found that the two were inversely related [ 911 ] . generally , weight - loss intervention studies have demonstrated that subjects who achieved larger weight losses when dieting are also those in whom the greatest increase in dietary restraint is noted [ 6 , 9 , 10 , 12 , 13 ] . in addition , the success of weight - loss interventions has also been associated to lower pre - weight - loss dietary restraint [ 10 , 14 ] . similarly , results from a longitudinal study have shown that a lower dietary restraint at baseline was associated with lower weight gain during a 6-year follow - up period . therefore in the long term , it is not clear whether it is preferable to have higher or lower dietary restraint for optimal body weight management . on the other hand , the association between disinhibition and bmi is more consistent as many studies have shown that higher dietary disinhibition is associated with higher bmi and a higher likelihood of weight gain over time [ 5 , 7 , 1618 ] . similarly , a positive correlation between hunger and bmi has been reported [ 5 , 17 ] . it has also been demonstrated that physical activity , apart from its impact on energy expenditure , could also influence energy intake . in fact , studies have suggested that physically active individuals are more likely to eat a healthy diet than sedentary individuals are . physically active individuals consume more fruits and vegetables and have higher intakes of fiber and calcium than sedentary individuals do [ 19 , 20 ] , which have been shown to favourably influence appetite and energy intake [ 21 , 22 ] . as well , physical activity can influence eating behaviour by improving satiety , by altering macronutrient preference , and by modulating the hedonic response to foods [ 2326 ] . considering the above - mentioned evidence suggesting that bmi is influenced by eating behaviour traits and that physical activity is generally associated with healthier food habits , the main objective of this paper was to investigate whether physical activity participation could influence the associations between eating behaviour traits and bmi in postmenopausal women . since it has been reported that exercise exerts some effects on eating patterns and that it has been shown to be more effective to regulate energy intake in restrained compared to nonrestrained eaters , we hypothesized that a negative association between dietary restraint and bmi would only be observed in women with higher physical activity participation . in addition , based on the fact that exercise does not act as a disinhibitor but rather increases the preference for low - fat foods and reduces the motivation to eat [ 28 , 29 ] , we also hypothesized that the positive associations between disinhibition and hunger with bmi would be attenuated with increased physical activity participation . the main objective of this cross - sectional study , conducted between 2000 and 2003 , was to determine the relative contribution of visceral adipose tissue and insulin resistance to the cardiovascular risk profile of postmenopausal women . a total of 386 women responded to the local newspapers of the qubec city metropolitan area . , one hundred ninety women were found to be eligible . among them , 69 dropped out of the study for personal reasons after having received a complete description of the research protocol . eight women who have either not totally completed the physical activity questionnaire or have answered less than 80% of each factor ( restraint , disinhibition and hunger ) were not included in the database . specifically , having answered to at least 17/21 , 13/16 , and 12/14 items for restraint , disinhibition and hunger were respectively needed to include the data in our analysis . in case of missing data , therefore , a total of 113 caucasian women aged between 46 and 67 years were included in the analyses for this paper . women were individually interviewed to evaluate whether they satisfied study 's inclusion criteria for age , postmenopausal status ( confirmed by absence of menses for at least 1 year and levels of follicle - stimulating hormone between 28 and 127 iul ) , absence of any hormone therapy ( ht ) , and other medication , except a stable dose of thyroxine that well - controlled a hypothyroidism 's diagnosis . at the time of inclusion , women had a stable weight for at least 2 months ( 2.5 kg ) , were not dieting , had no chronic diseases , and were not taking medication that could impact on the study outcome . this study was conducted according to the guidelines laid down in the declaration of helsinki , and all the procedures involving human subjects were approved by the universit laval medical ethics committee . written informed consent was obtained from all subjects / patients . body weight was measured to the nearest 0.1 kg using a calibrated weighing device including a tension gauge ( intertechnology inc . ) and a digital panel indicator ( beckman industrial series 600 ) . waist circumference was assessed in duplicate at the mid - distance between iliac crest and last rib margin with a flexible steel metric tape to the nearest 0.1 cm . eating behaviour traits were evaluated using the three - factor eating questionnaire , a 51-item validated questionnaire [ 3 , 33 ] . it assesses 3 factors and specific subscales that refer to cognitions and behaviours which have been reported to show good test dietary restraint is a conscious control of food intake to control body weight [ 35 ] . this factor can be divided into rigid dietary restraint ( dichotomous , all - or - nothing approach to eating , dieting , and weight ) and flexible dietary restraint ( gradual approach to eating , dieting , and weight ) . dietary disinhibition is characterized by an overconsumption of foods in response to a variety of stimuli ( e.g. , emotional stress ) associated with a loss of control on food intake [ 35 ] . it is further divided into three specific subscales : habitual susceptibility to disinhibition ( behaviours that may occur when circumstances predispose to recurrent disinhibition ) , emotional susceptibility to disinhibition ( disinhibition associated with negative affective states ) , and situational susceptibility to disinhibition ( disinhibition initiated by specific environmental cues ) . hunger represents food intake in response to feelings and perceptions of hunger . internal hunger ( hunger interpreted and regulated internally ) and external hunger ( triggered by external cues ) are the two specific subscales that can be derived from the hunger factor [ 4 , 34 ] . physical activity participation was defined using the 3-day activity diary record by bouchard et al . that was administered during two weekdays and one weekend day . each day ( 24 hours ) as previously described by major et al . , women reported the dominant activity that they were engaged in for each 15-minute period and indicated the corresponding number ( from 1 to 9 ) . if their specific activities were not included into the list , they were instructed to choose an activity with similar intensity . as an example , category 1 refers to activities of very low energy expenditure ( e.g. , sleeping and resting in bed ) , category 6 refers to leisure activities and sports in a recreational environment ( e.g. , golf , baseball , and volleyball ) , and category 9 refers to activities of very high energy expenditure ( e.g. , running ) . our study focused on mean daily energy expenditure from activities in categories 6 , 7 , 8 , and 9 ( ee69 ) , which have an energy cost > 1.2 kcalkg15 min ( > 4.8 mets ) ( i.e. , 1.2 , 1.4 , 1.5 , and 2 kcalkg15 min for categories 6 , 7 , 8 , and 9 , resp . ) . as previously described by major et al . , ee69 was calculated by multiplying the number of 15-minute periods of categories 6 to 9 by the approximate median energy cost of each category . as such , the following formula was used : ee69 = ( number of 15-minute periods of category 6 1.2 ) + ( number of 15-minute periods of category 7 1.4 ) + ( number of 15-minute periods of category 8 1.5 ) + ( number of 15-minute periods of category 9 2 ) . the result was then used to calculate the mean ee69 value for 3 days ( kcalkgday ) and was used for further analyses . the main limitation of this physical activity diary relates to the approximation of the energy cost since each categorical value is the approximate median amount of energy expended when engaged in the activities of the specific category . the 3-day activity diary record has nonetheless been validated , and results suggest that it represents an appropriate way to estimate mean daily energy expenditure and frequency of participation in activities from different categories . in addition , it has been suggested , in a study conducted in the same cohort , that women with higher physical activity participation had a better metabolic profile ( insulin sensitivity and lipid levels ) for a given level of visceral adipose tissue , which is concordant with the well - known beneficial effects of a higher physical activity participation on metabolic profile . dietary intakes were collected using a 3-day weighed food record , which was completed during two weekdays and one weekend day ( same days as the 3-day activity diary ) . guidelines for completing the food record were explained to participants by the study 's registered dietitian . the evaluation of nutrient intakes derived from the food record was performed using the nutrition data system for research software ( version 4.03 , developed by the nutrition coordination center , university of minnesota , minneapolis , mn , food and nutrient database 31 , released in november 2000 ) . all records were reviewed by the study 's registered dietician upon collection . in order to control for underreporting in our study population , subjects who reported an energy intake of less than 1 standard deviation from the mean reported energy intake were excluded from the dietary analysis ( 22 and 14 subjects in the lower and higher physical activity participation group , resp . ) . thus , the number of subjects included in the dietary analysis ( 42 and 35 subjects in the lower and higher physical activity participation group , resp . ) is different from the numbers included for the main outcome of the study ( 64 and 49 subjects in the lower and higher physical activity participation group , resp . ) . statistical analyses were performed with the use of spss software ( version 11.5 ; spss inc , chicago , il ) . the median value of ee69 ( 2 kcalkgday ) was used to form two groups : women with lower and higher physical activity participation . difference between the two groups for anthropometric variables , eating behaviour traits , and dietary intakes were assessed using student t - tests . pearson correlations were used to examine associations between bmi and eating behaviour traits within women characterized by lower and higher physical activity participation . bmi was compared between the four groups formed on the basis of the physical activity participation median and of the dietary restraint median ( 2 kcalkgday and a score of 9 , resp . ) , by using an analysis of variance ( anova ) , followed by tukey post hoc tests . the same analyses were performed with groups formed on the basis of the physical activity participation median and external hunger median ( 2 kcalkgday and a score of 2 , resp . ) . eating behaviour traits that were significantly correlated with bmi were used in multivariate linear regressions analyses ( i.e. , enter ) with bmi as a dependent variable . in addition , when a given tfeq factor and some of its subscales were significantly related to bmi , we chose to enter the subscales rather than the main factor in the multivariate model in order to avoid problems related to multicollinearity . participant characteristics ' from the 2 groups formed on the basis of physical activity participation ( according to the ee69 median value of 2 kcalkgday ) are shown in table 1 . participants with lower physical activity participation had significantly higher percentage of body fat ( 40.2 7.7 versus 37.4 7.1% , p < .05 ) and tended to have greater waist circumference ( 93.0 13.7 versus 88.4 12.7 cm , p = .07 ) . no significant differences in eating behaviour traits were noted between groups divided based on physical activity participation . with regards to dietary intakes , it was found , after removing underreporters from the sample , that women with lower physical activity participation consumed significantly more energy when compared to women with higher physical activity participation ( 1981.4 275.6 versus 1870.8 190.5 kcal , p < .05 ) . trends were also found for dietary cholesterol and fiber consumption and suggested a higher cholesterol ( 274.8 119.5 versus 227.8 85.9 mg , p = .06 ) and a lower fiber consumption ( 11.3 3.4 versus 12.5 2.6 g1000 kcal , p = .09 ) in women with lower physical activity participation when compared to women with higher physical activity participation . significant correlations were observed between eating behaviour traits and bmi in both groups of women separated on the basis of physical activity participation ( table 2 ) . in women with lower physical activity participation , flexible dietary restraint was negatively associated with bmi ( r = 0.24 , p < .05 ) while disinhibition ( r = 0.55 , p < .0001 ) and its subscales ( habitual ( r = 0.49 , p ( r = 0.35 , p < .005 ) ) as well as hunger ( r = 0.45 , p < .0001 ) and its subscales ( internal ( r = 0.41 , p < .0001 ) ) were all positively associated with bmi . in the group with higher physical activity participation , dietary restraint ( r = 0.54 , p < .0001 ) and its subscales ( flexible ( r = 0.55 , p < .0001 ) and rigid ( r = 0.37 , p < in contrast , disinhibition ( r = 0.42 , p < .005 ) , emotional ( r = 0.41 , p < .005 ) and situational susceptibility to disinhibition ( r = 0.30 , p < .05 ) as well as susceptibility to hunger ( r = 0.33 , p < .05 ) were all positively associated with bmi . similar results were obtained when percent body fat was correlated with eating behaviour traits ( results not shown ) . because correlations obtained with percentage of body fat were similar to those with bmi and that bmi represents an easily accessible proxy of adiposity , further analyses were conducted with bmi . comparisons of correlation coefficients for the associations between eating behaviour traits and bmi were performed between women with lower and higher physical activity participation . results showed significant differences in correlation coefficient for dietary restraint ( p = .02 ) and for external hunger ( p = .04 ) . for dietary restraint , a stronger correlation with bmi was found in women in the higher physical activity participation compared to those in the lower physical activity participation group ( r = 0.54 versus r = 0.16 , p = .02 ) . for external hunger , a stronger correlation was found in women from the lower physical activity participation group than in those from the higher physical activity participation group ( r = 0.49 versus r = 0.14 , p = .04 ) . trends were also found for flexible and rigid dietary restraint ( p < .06 and p < .07 , resp . ) whereas stronger correlations observed with bmi were found in the higher physical activity participation group . as such , analyses were performed with eating behaviour traits for which a significant difference in the strength of correlation with bmi was observed between women in the lower and higher physical activity participation groups ( i.e. , dietary restraint and external hunger ) . figure 1 shows the difference in the association between dietary restraint and bmi according to physical activity participation . among women with lower physical activity participation , bmi was not different between women with either lower ( mean : 29.3 1.0 kg / m ) or higher dietary restraint ( mean : 28.9 0.9 kg / m ) . on the other hand , among women with higher physical activity participation , bmi was significantly higher in women with lower dietary restraint ( mean : 30.3 1.7 kg / m ) when compared to women with higher dietary restraint ( mean : 25.5 0.5 kg / m ) . finally , it was also found that physical activity participation had an impact on the association between external hunger and bmi ( figure 2 ) . among women with lower physical activity participation , bmi was significantly lower in women with lower external hunger ( mean : 27.5 0.8 kg / m ) than in those with higher external hunger ( mean : 32.4 1.1 kg / m ) . no such differences were observed in women with higher physical activity participation ( mean : 27.2 1.3 and 28.6 1.1 kg / m for groups with lower and higher external hunger group , resp . ) . in order to investigate the relative and independent contribution of eating behaviour traits to the variability of bmi in both the lower and higher physical activity participation groups , multiple regression analysis ( i.e. , enter ) was performed . eating behaviour traits were included in the regression analyses if they were significant correlates of bmi . when both the subscales and the main tfeq factor were significant correlates , only the subscales were entered into the model . as such , emotional disinhibition and to a lesser amount external hunger were found to be independent contributors to bmi ( 35% of the variance after adjustment ) in women with lower physical activity participation ( table 3 ) while flexible dietary restraint , rigid dietary restraint , and to a lesser amount emotional disinhibition were the independent contributors to bmi ( 38% of the variance after adjustment ) in women with higher physical activity participation ( table 4 ) . the main objective of this paper was to investigate whether physical activity participation could interrelate with the associations between eating behaviour traits and bmi . our results suggest , for the first time , that dietary restraint is more strongly correlated with bmi in women with higher physical activity participation than in women with lower physical activity participation . moreover , flexible dietary restraint and rigid dietary restraint are independent predictors of bmi only in women with higher physical activity participation . our analyses also revealed that emotional disinhibition contributes to the variance in bmi in women with lower physical activity participation and only marginally in women with higher physical activity participation . finally , hunger , and more particularly external hunger , is strongly correlated with bmi in women with lower physical activity participation . it is well documented that both physical activity and dietary restraint impact body weight management . however , the influence of physical activity on the association between dietary restraint and weight has not been well established . our results show that postmenopausal women with higher physical activity participation and higher dietary restraint have a lower bmi when compared to women with higher physical activity participation and lower dietary restraint . this may be partially explained by the observation that women with higher physical activity participation seem to present healthier food habits ( lower cholesterol intakes and higher fber intake ) when compared to women with lower physical activity participation , and this , despite similar dietary restraint . these findings are concordant with other studies showing that women with higher physical activity participation are more likely to consume a healthy diet and to be characterized by healthier eating patterns [ 19 , 20 , 2325 ] . the observation that women who exercise seem to have the capacity to better regulate their appetite and that exercise can also possibly raise the perceived pleasantness of low - fat foods may partly explain why active women are more likely to chose a healthier diet , including foods with a low fat content [ 23 , 25 , 38 ] . another explanation to how physical activity participation could influence the association between dietary restraint and bmi relates to the association between dietary restraint and disinhibition . in fact , some studies have underlined the heterogeneity of the association between dietary restraint and disinhibition , with results reporting negative [ 5 , 39 , 40 ] , positive [ 39 , 40 ] , or no association [ 5 , 41 , 42 ] between these variables . our results have shown an association between dietary restraint and disinhibition in women with higher physical activity participation while no significant correlation between these variables was found for women with lower physical activity participation ( r = 0.29 , p < .05 ; r = 0.16 , p = ns ; higher and lower physical activity participation , resp . ) . this observation is also strengthened by the fact that disinhibition , in the higher physical activity participation group , was significantly lower in women who displayed a higher dietary restraint when compared to women with a lower dietary restraint ( 4.9 3.1 versus 7.2 4.4 , p = .04 , resp . ) . no such significant differences in disinhibition were noted when women with either lower or higher dietary restraints were compared with women in the lower physical activity participation groups . since a lower disinhibition level has been reported in many studies to be predictive of a lower bmi [ 5 , 7 , 1618 ] , the inverse association between dietary restraint and disinhibition among women with a higher physical activity participation could explain , at least in part , the fact that a higher dietary restraint is associated to a lower bmi among this group of women . our results also showed that although flexible dietary restraint and rigid dietary restraint were both significant predictors of bmi , flexible dietary restraint was the strongest predictor , explaining 34% of the variance in bmi among women with higher physical activity participation . these results are concordant with previous studies showing that flexible dietary restraint is a better predictor of lower bmi than rigid dietary restraint . for example , longitudinal studies showed that changes in flexible dietary restraint , but not changes in rigid dietary restraint , correlated negatively with changes in body weight [ 8 , 15 ] . it has been previously shown that a higher disinhibition level predicts higher bmi and higher likelihood of weight gain [ 5 , 7 , 1618 ] . our results add to this literature by showing that in women with higher physical activity participation , disinhibition does not predict the variability in bmi ( p = .06 ) while it predicted 39% of bmi variability among women with lower physical activity participation ( p < .005 ) . therefore , in women with higher physical activity participation , disinhibition does not seem to have as much of an impact on bmi when dietary restraint is taken into account . in fact , it can be hypothesized that after a disinhibition episode , women with a higher physical activity participation are able to respond by reducing their energy intake over the course of the following meals in order to minimize the impact of disinhibition on energy balance and/or increase their physical activity . among women with lower physical activity participation , a higher bmi was noted in women characterized by higher external hunger when compared to those with lower external hunger . in contrast , no difference in bmi was observed according to external hunger value among women with higher physical activity participation . interestingly , we found that among women with lower physical activity participation , external hunger was positively associated with energy intake ( r = 0.36 ; p < .005 ) , the percent of energy from dietary lipids ( r = 0.29 ; p < .05 ) , and cholesterol intake ( r = 0.32 ; p < .01 ) . no such associations were noted in women with higher physical activity participation . therefore , women with lower physical activity participation and higher external hunger , increased energy and dietary fat intake could explain , at least in part , their increased bmi . the reasons why external hunger is not associated with dietary factors potentially leading to positive energy balance among women with higher physical activity participation will have to be further elucidated . our findings are limited to a small population of postmenopausal women and should thus be interpreted accordingly . in addition , even if it remains that the 3-day activity diary record has been previously validated and that the use of self - reported physical activity and dietary data are , to some extent , a limitation . furthermore , the cross - sectional nature of this study makes it difficult to underline the possible interactions between behaviour , environment , and genes . thus , it does not exclude the possibility that other variables might influence interactions between eating behaviour traits and bmi . because of the cross - sectional nature of this study , it is obvious that we can not allude to any causality for physical activity participation on the association between eating behaviour traits and bmi . it is nonetheless tempting to speculate that depending on their level of physical activity participation , some individuals could react differently to eating behaviour interventions aimed at preventing weight gain or inducing weight loss . for example , it might be suggested that increasing dietary restraint might be a more efficient approach to lose weight or to avoid weight gain among women with higher physical activity participation than in those with lower physical activity participation . of course , this remains to be tested in well - designed weight management interventions .

available data reveals inconsistent relationships between eating behaviour traits and markers of adiposity level . it is thus relevant to investigate whether other factors also need to be considered when interpreting the relationship between eating behaviour traits and adiposity . the objective of this cross - sectional study was thus to examine whether the associations between variables of the three - factor eating questionnaire ( tfeq ) and adiposity are influenced by the level of physical activity participation . information from the tfeq and physical activity was obtained from 113 postmenopausal women ( 56.7 4.2 years ; 28.5 5.9 kg / m2 ) . bmi was compared between four groups formed on the basis of the physical activity participation and eating behaviour traits medians . in groups of women with higher physical activity participation , bmi was significantly lower in women who presented higher dietary restraint when compared to women who had lower dietary restraint ( 25.5 0.5 versus 30.3 1.7 kg / m2 , p < .05 ) . in addition , among women with lower physical activity participation , bmi was significantly lower in women presenting a lower external hunger than in those with a higher external hunger ( 27.5 0.8 versus 32.4 1.1 kg / m2 , p < .001 ) . our results suggest that physical activity participation should also be taken into account when interpreting the relationship between adiposity and eating behaviour traits .

1. Introduction 2. Experimental Methods 3. Results 4. Discussion

PMC2238924

orthologs are defined as genes in different species deriving from a single gene in the last common ancestor ( 1 ) , and are therefore likely to have the same function . if an ortholog undergoes duplication in one species , the copies are referred to as inparalogs ( 2 ) . inparalogs are by definition co - orthologs to one or more orthologs in another species . in contrast , two genes deriving from a duplication that predated the speciation event between the species are referred to as outparalogs . the inparanoid program was developed to identify clusters of inparalogs while avoiding inclusion of outparalogs . inparanoid is one of the first comprehensive ortholog databases ( 3,4 ) , but nowadays more than 15 different ortholog databases exist ( 5 ) . a reason for the multitude of ortholog databases is that different research questions have different needs . for instance , the cogs database ( 6 ) contains very large clusters of orthologs that often contain outparalogs ( 7 ) . at the other extreme , the homologene database ( 8) often places inparalogs in different clusters . however , the average user is normally interested in simply finding all orthologs in species y to a gene in species x , including all inparalogs but excluding outparalogs . two benchmarks have recently been published that try to objectively assess the quality of different ortholog databases ( 9,10 ) . in both these tests , which look either at accuracy of functional annotation or at inferred accuracy , this suggests that inparanoid is successful at balancing the false - negative and false - positive rate , and is appropriate as a general - purpose orthology tool . this release contains a number of fixes to minor bugs that could lead to incorrect cluster merging or bootstrap values . the website has been completely reconstructed and has new front- and back - ends , yet looks very similar to the old site . the new design makes it much faster for the user , and allows easier updating of the system . with the new back - end the data was gathered from three different sources : ensembl , ncbi and model organism databases ( mods ) . we only considered eukaryotic genomes sequenced to a coverage greater than 6x , with < 1% unknown amino acids ( x in the protein sequences ) . most mod data was packaged and uploaded by the staffs at tair , wormbase , flybase , zfin , dictybase , sgd and mgi to us directly , but three mods were downloaded from their repositories . before running inparanoid , each proteome was made non - redundant by keeping only the longest transcript from each gene . if this is not done first , different transcripts from the same gene can end up in different clusters if they exist in more than one species . together , we have formed an informal consortium of mods that want to cross - reference each other using orthologs from inparanoid . we particularly welcome this system as it allows us to use the most complete and recent set of proteins for each organism , and ensures that we use identifiers that work in the mods so that web links to proteins are valid . we hope that more mods will join in and provide their proteomes in a new and robust xml format that will be introduced for the next release . from ensembl , data was obtained for aedes aegypti ( transcripts for 15 419 genes ) , anopheles gambiae ( 13 277 ) , apis mellifera ( 13 448 ) , bos taurus ( 22 280 ) , canis familiaris ( 19 314 ) , ciona intestinalis ( 14 278 ) , gallus gallus ( 16 715 ) , gasterosteus aculeatus ( 20 879 ) , homo sapiens ( 22 983 ) , macaca mulatta ( 22 045 ) , monodelphis domestica ( 19 597 ) , pan troglodytes ( 20 982 ) , rattus norvegicus ( 23 299 ) , takifugu rubripes ( 22 008 ) , tetraodon nigroviridis ( 28 005 ) and xenopus tropicalis ( 18 473 ) . apis mellifera was taken from ensembl release 38 and all other proteomes from release 43 . from ncbi , we obtained candida glabrata ( 5192 ) , cryptococcus neoformans ( 6487 ) , debaromyces hansenii ( 6318 ) , entamoeba histolytica ( 9772 ) , escherichia coli k12 ( 4243 ) , entamoeba histolytica ( 9772 ) , kluyveromyces lactis ( 5336 ) , yarrowia lipolytica ( 6544 ) . the mods uploaded proteomes for arabidopsis thaliana ( 26 819 ) , caenorhabditis briggsae ( 19 334 ) , caenorhabditis elegans ( 20 084 ) , caenorhabditis remanei ( 25 595 ) , danio rerio , ( 12 303 ) , dictyostelium discoideum ( 13 523 ) , drosophila melanogaster ( 13 854 ) , mus musculus ( 23 132 ) , saccharomyces cerevisiae ( 5792 ) . we obtained from other mods oryza sativa ( 77 853 ) ( from http://www.gramene.org ) , drosophila pseudoobscura ( 9871 ) ( from http://www.flybase.org ) , and schizosaccharomyces pombe ( 5003 ) ( http://www.sanger.ac.uk ) . blast comparisons using these datasets were performed between each pair of species , involving four whole proteome runs per species pair ( normal runs both ways plus two self - self runs ) . for the 35 proteomes this amounts to 595 species pairs , requiring 1225 whole - proteome blast searches . the pairwise blast results were used as the input for the inparanoid ortholog clustering procedure ( 3 ) . the output from inparanoid 6 is available as xml , sql , html and native format for downloading at the inparanoid homepage , and is searchable via the web interface . ncbi blast comparisons using these datasets were performed between each pair of species , involving four whole proteome runs per species pair ( normal runs both ways plus two self - self runs ) . for the 35 proteomes this amounts to 595 species pairs , requiring 1225 whole - proteome blast searches . the pairwise blast results were used as the input for the inparanoid ortholog clustering procedure ( 3 ) . the output from inparanoid 6 is available as xml , sql , html and native format for downloading at the inparanoid homepage , and is searchable via the web interface . the 35 species present in the inparanoid database result in 595 pairwise ortholog lists . the information in these lists was used to generate a phylogenetic tree that reflects the level of orthology between the different species . we calculated the orthology distance from species a to b , dab , by and used the average orthology distances ( dab + dba)/2 to construct a upgma tree , shown in figure 1 . , it agrees with the standard taxonomic species tree , but we noted a few exceptions . opossum ( m. domesticus ) , a marsupial mammal , is clustered together with placental mammals , and the zebrafish d. rerio clustered as an outgroup to the land animals rather than together with other fish . the latter anomaly is very minor as all fish are still neighbors in the tree , but the placement of opossum is surprising . if this placement is correct , then marsupials could have evolved from a particular lineage of placental mammals . k. lactis , s. cerevisiae and d. hansenii are clustered together , while c. glabrata is placed outside this group . these are arranged differently in the orthophylogram in that c. glabrata has traded place with d. hansenii , which now is placed as an outgroup to k. lactis , s. cerevisiae and c. glabrata . surprisingly , the green plants are placed as a subgroup among single - cell organisms , next to the fungal group . for instance , on average 91.2% of the proteins in h. sapiens and p. troglodytes are orthologous . the tree topology generally corresponds to the standard taxonomy , but a few exceptions were noted ( see text ) . for instance , on average 91.2% of the proteins in h. sapiens and p. troglodytes are orthologous . the tree topology generally corresponds to the standard taxonomy , but a few exceptions were noted ( see text ) . it is worth noting that on average only 91.2% of the proteins in h. sapiens and chimpanzee p. troglodytes are orthologous . this is surprisingly low since the genome - wide nucleotide divergence between human and chimpanzee is estimated to only 1.23% ( 12 ) . the much higher difference observed for orthologs is not due to unique proteins in either proteome , as the fraction of homologs reported by inparanoid is 96.7% for human and 99.3% for chimpanzee . this is , however , often caused by incomplete sequencing or errors in gene annotation . the average number of inparalogs per cluster ranges from 1.001 ( in drosophila pseudoobscura when compared to d. melanogaster ) to 7.160 ( in o. sativa when compared to d. rerio ) . the overall mean number of inparalogs per species was 1.54 , and the median was 1.25 . the highly duplicated genome of o. sativa is responsible for all average cluster sizes of four , and generates a separate peak in the distribution around five . in fact , o. sativa had on average more than four inparalogs per ortholog group when compared to every other non - plant species . it is surprising that the average number of inparalogs in o. sativa was so high when compared with d. rerio ; when compared with d. rerio 's phylogenetic neighbors the number was only around five . although the rice proteome clearly contains the largest number of genes , our figures are probably somewhat overestimated . evidence for this is that we were not able to find shared gene identifiers between any rice proteins in the mod . this problem will be resolved in the future by collaborating directly with the rice mod staff to get a better - annotated rice proteome . figure 2.histogram of average number of inparalogs / cluster per species for all species species comparisons in inparanoid 6 . the peak around five inparalogs / cluster is entirely caused by o. sativa , rice . histogram of average number of inparalogs / cluster per species for all species species comparisons in inparanoid 6 . the peak around five inparalogs / cluster is entirely caused by o. sativa , rice . the inparanoid database is freely available at http://inparanoid.sbc.su.se . in addition to the data which is available to search / browse using the web interface , fasta files containing all proteins , protein description files , ortholog tables in raw , sql and xml format are available for each pairwise inparanoid analysis .

the inparanoid eukaryotic ortholog database ( http://inparanoid.sbc.su.se/ ) has been updated to version 6 and is now based on 35 species . we collected all available complete eukaryotic proteomes and escherichia coli , and calculated ortholog groups for all 595 species pairs using the inparanoid program . this resulted in 2 642 187 pairwise ortholog groups in total . the orthology - based species relations are presented in an orthophylogram . inparanoid clusters contain one or more orthologs from each of the two species . multiple orthologs in the same species , i.e. inparalogs , result from gene duplications after the species divergence . a new inparanoid website has been developed which is optimized for speed both for users and for updating the system . the xml output format has been improved for efficient processing of the inparanoid ortholog clusters .

INTRODUCTION DATA AND IMPLEMENTATION InParanoid clustering INPARANOID CONTENT DATA AVAILABILITY

PMC3496346

understanding and exploiting the chemical driving forces responsible for tight and specific molecular interactions remains a fundamental challenge in science and engineering . in practical molecular design applications such as structure - based drug design , a lead compound often serves as a starting point for optimization , in which the compound ( or ligand ) is rationally modified in the context of the target protein ( or receptor ) , to improve its pharmacological parameters , including potency and selectivity . geometrical and physicochemical complementarity is often required for tight binding , and the quality of this complementarity is generally reflected in the van der waals and electrostatic contributions to binding . however , whereas the van der waals binding contribution is generally strongly favorable , the net electrostatic contribution is often neutral or even somewhat unfavorable . ligand electrostatic interactions are acquired through the loss of their individual interactions with solvent in the unbound state , which results in a desolvation penalty . the task of ligand optimization often involves incremental improvement of the shape or electrostatic complementarity , although other sources can also be exploited . charge optimization theory has been developed in the context of linear response theories for the study of electrostatic contributions to binding affinity . the fundamental observation is that the ligand contribution to the binding affinity can be expressed in a simple mathematical form comprising a desolvation penalty that goes as the square of the ligand charge distribution and a generally favorable screened intermolecular interaction term that is linear in the ligand charge distribution . the tradeoff of these terms and their different dependencies on the ligand charge distribution result in an optimum charge distribution corresponding to the most favorable electrostatic contribution to binding . initially , the geometry of both the free ligand and the bound complex were treated as spherical , and the ligand charge distribution was expressed as a set of multipoles in order to solve the problem . nonspherical geometries and alternative basis sets for ligand charge distributions were then addressed , and the method was extended to exact molecular shapes . a framework for optimizing electrostatic specificity calculation of the electrostatic potentials is generally done by solving the linearized poisson boltzmann ( lpb ) equation , but other forms of linear response theory are applicable . a series of algorithmic advances has been made to accelerate the calculation and produce more - accurate lpb solutions . charge optimization theory has been broadly applied to probe or design ligands in various molecular systems . kangas and tidor applied charge optimization theory to study the binding between the bacillus subtilis chorismate mutase and an endo - oxabicyclic transition - state analogue . they found that , although the inhibitor showed very good electrostatic complementarity to the enzyme active site , a carboxylate group lost more in desolvation penalty than it gained in interactions with the enzyme ; the calculations suggested that substitution with a nitro group would improve the binding affinity . mandal and hilvert synthesized the new compound and found , experimentally , a 1.7-kcal / mol improvement in binding affinity in a context corresponding to the calculational study , thus identifying the most potent known inhibitor of this enzyme . other applications include e. coli glutaminyl - trna synthetase binding to its cognate substrates , protein inhibitors of hiv-1 cell entry , the interface between protein kinases and their ligands , small - molecule influenza neuraminidase inhibitors , and the celecoxib ligand binding independently to cox2 and caii . recently , charge optimization and protein design together identified tighter binding peptides to hiv-1 protease that were studied experimentally . binding specificity optimization probed ligand binding in the model system of hiv protease , other proteases , and their inhibitors . the charge optimization approaches described above are based on considering the ligand to remain rigid as it binds to a receptor . a comprehensive theory that includes ligand conformational change on binding has been lacking in the field . indeed , as one of the authors has shown , a direct replacement of the unbound ligand structure in charge optimization produced charges that strongly stabilized the bound ( preconformed ) over the unbound conformation , which is physically implausible . in this study , we explore the electrostatic complementarity of an induced - fit ligand to its receptor , and we generalize charge optimization theory to such cases . for our purposes , an induced - fit ligand has one conformation in the unbound state that may or may not be the same as the preconformed structure in the bound state . the origin of the previous unphysical results when the rigid theory was applied to flexible ligands is analyzed , and new methodology is developed and applied to the binding affinity optimization problem for a designed hiv-1 protease inhibitor , mit-2-kb-98 . it generates an optimum partial atomic charge distribution that can be different from that with the rigid - ligand assumption . the key tenet of the new theory is the constraint that the preconformed ligand can not be lower in free energy than the unbound conformation ( otherwise , it would be the unbound state ) . ( theory ) , we briefly introduce rigid - ligand charge optimization theory and then describe our treatment of the induced - fit charge optimization problem , in which the ligand adopts an unbound conformation potentially different from the bound . in our formulation , the unbound ligand is either predefined or selected from a set of candidate conformers . the latter framing is useful for future extensions of the theory in which the unbound state is treated as an ensemble representing a population distribution . whereas nonelectrostatic binding contributions cancel for rigid charge optimization , they can persist in the induced - fit theory developed here . section 3 ( methods ) introduces the test system of hiv-1 protease and its designed inhibitor , as well as methods for calculating continuum electrostatic potentials and nonelectrostatic energies , and approaches to solving the optimization problem . in section 4 ( results ) , we analyze the decomposed thermodynamic consequences of the derived results and compare them to those with the rigid - ligand charge optimization approach . molecular binding in an aqueous solvent can be usefully viewed not as an association reaction , in which only new intermolecular interactions are introduced between receptor and ligand , but rather as an exchange reaction in which some receptor solvent and ligand solvent interactions present in the unbound state are lost to accommodate the gain of receptor ligand interactions in the bound complex . the net effect of the exchange nature of binding is potentially interesting and nonintuitive for electrostatics , and leads to charge optimization theory . here , we first review the theory for the binding of a rigid ligand to form a complex and then extend the theory to treat induced - fit binding , in each case solving for the ligand charge distribution that leads to the most favorable binding free energy . consider the binding of receptor ( r ) and ligand ( l ) to form complex ( c ) in an aqueous environment . the charge distribution is modeled as a set of point charges qi at atomic centers ri . the macromolecules are treated as low dielectric volumes defined by their molecular shapes that are surrounded by high dielectric solvent with ions at physiological concentration . the electrostatic binding free energy of the complex can be written as1where gxes is the electrostatic free energy of molecule x , which has a particularly convenient form in any linear response theory . here , we illustrate using solutions to the linearized poisson boltzmann ( lpb ) equation , without loss of generality:2equation 2 inter - relates positional maps of electrostatic potential , charge density distribution , and dielectric constant ( or permittivity ) , where (r ) = [ 8ei(r)/(kt ) ] is the inverse debye length with i the ionic strength , e the electron charge magnitude , k the boltzmann constant , and t the absolute temperature . the equation is generally solved for the electrostatic potential . in linear response theory , the electrostatic free energy of a molecule x is given by3where the summation runs over the partial atomic charges qj located at positions rj . in the lpb model , individual point charges act independently and obey superposition , so their contributions can be calculated separately and summed,4where i(rj ) ( the electrostatic potential at position rj due to a unit point charge at ri ) can be calculated by solving the lpb equation ( eq 2 ) . we note that procedures are generally implemented to avoid the coulombic singularity for i = j , but we will leave the singularity in the equations here . by plugging this equation into eq 3 , one can write5after introducing a matrix x for molecule x ( x(i , j ) = ( 1/2)i(rj ) ) and splitting it into solvent reaction field and coulombic terms , as well as expressing the partial atomic charges of x as a vector qx = ( q1,q2,q3, ) , one can rewrite eq 5 as6combining eqs 1 and 6 , the electrostatic binding free energy of a complex can be expressed as7denote the matrices csolv and ccoul as cy and consider their structure . each element cy(i , j ) = ( 1/2)i(rj ) is half the potential ( solvent reaction field or coulombic , depending on y ) at point rj due to a unit charge at ri . the locations ri and rj can both be in the ligand , both be in the receptor , or one in each . it is useful to indicate these three cases by representing cy in block form as8 in this format , rcy is a square matrix of dimension nr ( the number of receptor partial atomic charges ) giving half the potential at partial atomic charge locations in the receptor due to the presence of other individual receptor unit charges , and lcy is the same for the ligand . ccy indicates a half - potential at a ligand atom due to a unit charge on a receptor atom and is not generally square . because of reciprocity , cy , rcy , and lcy are all symmetric , and ccy and ( ccy ) are indeed transposes of each other . for all cases , the subscript c indicates that the potentials are taken in the protein complex . while this does not affect the coulombic potentials ( taken in uniform low dielectric environment ) , it is important , because it defines the molecular boundary for the reaction field potentials . the first two terms of eq 7representing the electrostatic free energy of the complex then can be rewritten as9the factors of 2 in the l similarly , for the unbound receptor or ligand , the matrices ry and ly can be represented as rxy and lxy , respectively , which give the potential at a receptor or ligand atom generated by atomic charges in state x ( x = u for the unbound state and x = c for the bound complex ) . therefore , the electrostatic contribution to the binding free energy in eq 1 can be rewritten as10 when both the receptor and the ligand are regarded as rigid molecules with no conformational change upon binding ( u = c for both molecules ) , the intramolecular coulombic terms cancel ( lccoul = lucoul and rccoul = rucoul ) . moreover the matrices l and r are expected to be positive semidefinite , because desolvation should represent a penalty and symmetric due to reciprocity . in this rigid docking mode with no conformational change , eq 10 can be simplified to11where rt = 2qrtcc = 2qrt(ccsolv + cccoul ) is the screened electrostatic potential ( including both the solvent reaction field and coulombic terms ) at the ligand atoms generated by receptor atomic charges qr . also , we have renamed gbindes to gdockes to describe the rigid case for convenience later . of the three terms in eq 11 , ligand desolvation is quadratic in the ligand charge distribution ql,12the intermolecular screened electrostatic interaction is linear in ql,13and receptor desolvation is a constant , independent of ql:14thus , the electrostatic binding free energy for rigid molecules ( gdockes ) is a paraboloid in the space of ql . moreover , there is a unique global optimum qlopt = lcctqr that balances the unfavorable ligand desolvation penalty and the favorable intermolecular interactions , and gives the minimum electrostatic binding free energy . kangas and tidor showed that this minimum value is upper - bounded by zero , and thus the optimized ligand charge distribution leads to favorable electrostatic binding free energy under certain conditions . natural proteins seem to use this type of optimization to achieve tight binding : the tight - binding inhibitor barstar was found to be electrostatically optimized to its partner barnase , as described by this charge optimization theory . in this extension of charge optimization theory , the unbound ligand is treated as a set of individual conformational candidates , one of which is selected by the optimization as a single unbound state conformation . in the current work , of these two simplifications , the former has no effect on ligand charge optimization , because the unbound receptor contribution to the binding free energy is a constant for all ligand charge distributions considered ; multiconformational effects for the bound complex could be significant and will be treated in future work . also considered in future work are ensemble treatments in which each state is represented as a boltzmann distribution of conformations rather than a single conformation . in this framework , the binding free energy can be deconstructed as the sum of two terms as shown in the blue row of the thermodynamic pathway shown in figure 1 . in the first step , the unbound ligand changes conformation to the preconformed ligand structure ( i.e. , that adopted by the ligand in the bound complex ) ; the free energy change for this step is termed the preconformation contribution gpre . in the second step , the preconformed ligand and receptor dock rigidly to form the bound complex ( as modeled in the rigid - ligand charge optimization problem above ) , and the free - energy change is termed the docking contribution gdock . thus , the total binding affinity is represented as the sum of two terms corresponding to the two processes:15each term is expressed as a summation of electrostatic and nonelectrostatic contributions.1617here , the nonelectrostatic terms include covalent energy terms ( bonds , angles , torsions , etc . ) , intramolecular and intermolecular van der waals interactions , and nonpolar solvation interactions . the process of induced - fit binding of a ligand to its receptor . binding is decomposed into the two steps of preconformation and docking . the ligand has two conformations , unbound ( u ) and preconformed ( p ) , which can be the same . calculation of the binding free energy is indicated schematically , using terms from eqs 20 and 21 . the blue row indicates solvated molecules and the upper row indicates calculations in uniform dielectric . in the preconformation step , the ligand is solvated in an aqueous environment alone and changes its conformation from the unbound ( u ) to the preconformed ( p ) structure . we stress that even for the preconformed structure , the ligand is free in solution . this is equivalent to a special case of eq 10 , in which the receptor is not present . therefore , the electrostatic contribution to the change in free energy ( gprees ) can be expressed as the sum of a solvent reaction field term ( gpresolv ) and a coulombic term ( gprecoul ) , given by18the nonelectrostatic contribution to the change in free energy is19where exnon - es denotes the nonelectrostatic contribution to the free energy for the ligand in a given conformation ( x ) . in the docking step therefore,20and21equations 20 and 21 can be interpreted from the thermodynamic pathway of figure 1 . gpre is equal to the difference in free energy of the ligand in two conformations ( u and p ) . in a given conformation x , the free energy of the ligand includes the cost of assembling the charge distribution ql in a uniform low dielectric ( qltlxcoulql ) and that of moving it to high - dielectric solvent ( qltlxsolvql ) , as well as the nonpolar contributions to the chemical potential ( exnon - es ) . in the original formulation of charge optimization theory , the binding reaction was treated as the rigid docking of preconformed ligand and receptor ; with no ligand conformational change ( gpre = 0 for that case ) . here , , we permit the unbound ligand to adopt one conformation that may be the same or different from the bound ( preconformed ) ligand conformation . charge optimization in the current framing requires optimizing the binding free energy with respect to the ligand charge distribution . in the formulation here , a set of candidate unbound ligand conformations is available , and optimization selects an unbound conformation and charge distribution for the ligand that optimizes binding free energy . one physical constraint that we explore in this study is that the conformation assigned as the ligand unbound state should correspond to the lowest energy conformation in the set of candidate conformations when evaluated with the optimized charge distribution . said another way , the unbound structure is constrained to be the ground unbound state . to carry out the desired optimization , we construct a variational binding free energy expression that includes all terms that depend on the ligand charge distribution , either explicitly or implicitly . all terms that depend on the unbound ligand conformation must be kept in the optimization , because they contribute to defining the unbound ligand ground - state energy . here , those include all terms in gpre except epnon - es , and , for simplicity , we keep all terms of gpre in our optimization function . the receptor electrostatic desolvation penalty and the nonelectrostatic contribution to gdock are both independent of ql and , therefore , have been removed to construct the optimization function gtotal , which can be written as22 when the unbound ligand conformation is predefined , the optimization task involves the following:23subject towhere qlk denotes the kth element of ql ( i.e. , the partial atomic charge for atom k of the ligand ) . the first constraint limits the magnitude of any single charge to qmax ( generally 0.85 units of the electron charge magnitude ) , and the second fixes the total charge of the ligand at qtotal ( usually an integer ) . if the unbound and bound ligand conformations are the same , this reduces to the original charge optimization approach ; if the two ligand conformations differ , this corresponds to the treatment of gilson . when , instead , the unbound ligand conformation is selected from a set of candidates , the optimization problem is somewhat different . the optimized ligand charge distribution qlopt must be selected such that ( 1 ) gtotal is minimized and ( 2 ) the chosen unbound ligand conformation has the lowest free energy of all conformations in the unbound set with qlopt applied . the second requirement implements the physical constraint that the conformation claimed for the unbound state be the lowest free energy of those considered , which we show avoids the pathological results observed when rigid theory was applied directly to the nonrigid case . denoting the set of unbound candidates by u and indexing each member by i , we write the following mathematical programming problem:24subject to note that z is not smooth in ql ( i.e. , z has discontinuous derivatives ) , because of the function min( ) . the lack of smoothness presents a fundamental challenge in solving the optimization problem and is highly undesirable in practice . thus , we reformulate the problem into the following smooth one with the same optimum solution , using a standard transformation technique that replaces the explicit min function to define z with an implicit one,25subject to this formulation is general , in that it includes the special cases of rigid ligand ( u containing only the preconformed , bound state ) and a two - state induced - fit ligand ( u containing only an unbound state different than the preconformed , bound state ) . the formulation is useful in that it can be extended to treat conformational ensembles representing population distributions rather than unique structures for individual states . also , note that , although the presentation here is in terms of a basis of point charges , the theory is readily applicable to other bases . the crystal structure of hiv-1 protease alone was extracted from its bound complex with the inhibitor darunavir ( pdb i d 1t3r ) and prepared as described by altman et al . with both catalytic residues ( asp 25 ) deprotonated . three conserved water molecules were retained , including the so - called flap water molecule hydrogen atoms were added to the complex with the hbuild module of the computer program package charmm . parameters for mit-2-kb-98 were assigned from the charmm22 set except for the partial atomic charges , which were derived here . fock ( rhf ) 3 - 21 g level as implemented in the program gaussian 03 . partial atomic charges were calculated for the new geometry by restrained electrostatic potential ( resp ) fitting to rhf/6 - 31 g * potentials . a set of 1000 unbound mit-2-kb-98 conformations was computed from a 30 dihedral grid using dead - end elimination ( dee ) and a * to select the 1000 lowest energy structures different from the preconformed design structure . this unbound conformer set covered the lowest 1.5 kcal / mol in free energy and over 92% of the boltzmann distribution of all the enumerated states . the energy function used was the gas - phase energy with a dielectric constant equal to 4 and without nonbond cutoffs . these structures were indexed by rank in energy from 1 ( lowest ) to 1000 ( highest ) , and , for convenience , the preconformed structure was given an index of 0 . solvation potential matrices were computed with continuum electrostatic calculations implemented in a locally modified version of the program delphi . calculations were performed for the preconformed ligand in the free state and complexed with the protein ( lpsolv lu,0solv and l ) , as well as for each of the other 1000 members of the unbound conformer set alone ( lu , isolv , i = 1 , ... , 1000 ) . details of the calculations were as given in the work of altman et al . partial atomic charges for hiv-1 protease and atomic radii of both the protein and the ligand were obtained from the parse parameter set . the dielectric constant was set to 4 for the molecular interior and 80 for the exterior , with the dielectric boundary represented by the molecular surface computed with a 1.4- probe . a 2.0- ion - excluding stern layer surrounded all molecules , and a bulk ionic strength of 0.145 m was employed . linearized poisson boltzmann ( lpb ) calculations were performed on a 129 129 129 grid with focusing boundary conditions ( successively 23% , 92% , and 184% fill ) . the intramolecular coulombic electrostatic potential matrices lu , icoul were calculated directly from coulomb s law ; 12 and 13 interactions were excluded and 14 interactions were scaled by a factor of 0.50 , which are consistent with the charmm22 treatment . charmm was used with the charmm22 parameter set to evaluate the nonelectrostatic energies eu , inon - es for the 1001 members of the set of unbound candidates , including internal energy terms ( bond , angle , dihedral , improper , and urey bradley ) and van der waals ( excluding the 12 and 13 interactions , and with special treatment of 14 interactions ; without nonbonded smoothing or truncation ) . the nonpolar contribution to the hydration free energy the induced - fit ligand charge optimization problem formulated in eq 23 is not guaranteed to be convex , which implies possible multiple local minima . in practice , there is no guarantee for a generic solver to determine the global minimum of a nonconvex optimization problem . in this study , we used multistart trajectories with a local minimizer . we chose conopt3 as the local minimizer for its numerical efficiency , as available through gams . conopt3 is a nonlinear optimization solver based on generic reduced gradient algorithms . for each problem , 100 starting ligand charge distributions were randomly generated by uniformly sampling in the feasible space ( the intersection of a hypercube defined by |qlk| qmax , k = 1 , ... , natom and a hyperplane by k=1natomqlk = qtotal ) . for this purpose , we adopted an algorithm decomposing the space into different types of simplexes and sampled within and across these simplexes uniformly . the solution was chosen as that with the lowest value of the goal function from the multistart set . results are presented for induced - fit charge optimization on a previously designed ligand binding to the enzyme hiv-1 protease . the ligand , mit-2-kb-98 , has an ( r)-(hydroxyethylamino)sulfonamide scaffold functionalized to make favorable interactions in substrate recognition subsites ( see figure 2b ) . it has a total of 13 rotatable torsion angles ( neglecting the two methyl groups and the amide bond ) , which were varied to create the set of unbound ligand candidates ( see the methods section ) . two different but related schemes were used here . for each , a collection of 1001 different conformations of the ligand mit-2-kb-98 was used to represent conformational candidates for the unbound ligand . the structure of the bound complex with the receptor hiv-1 protease was taken as the design structure from the study in which this ligand was designed , synthesized , and assayed . of the 1001 unbound ligand conformers , conformer number 0 corresponds exactly to the preconformed ( bound ) ligand structure , whereas conformations 11000 correspond to ordered low - energy rotameric variants of increasing energy with dihedral angles selected from a 30 grid , identified with the a * algorithm . in one of the approaches , termed single - conformer unbound state , each of the 1001 unbound ligand conformers was considered in turn to individually be the unbound state . with conformer 0 treated as the unbound state , this corresponded to rigid - ligand charge optimization . with conformer 11000 as the unbound state , this corresponded to one form of induced - fit charge optimization . in the second approach , termed dominant - conformer unbound state , an additional constraint was placed on the ligand . namely , the conformation selected as unbound state was required to be of the lowest free energy in the set of unbound structure candidates , consistent with physical principles . that is , the unbound state is the ground state of the candidate set . in the work presented here , the unbound candidate set was grown by progressively adding members to examine size effects . this second form of induced - fit optimization is the main contribution of the current work . ( a ) designed inhibitor mit-2-kb-98 ( stick representation ; green ) in model complex with hiv-1 protease ( cartoon representation ; cyan ) , with the three retained water molecules ( line representation ) . ( b ) chemical structure of mit-2-kb-98 . to facilitate analysis of the results , the overall binding free energy gtotal is considered as the sum of two processes : a preconformation step , in which the unbound ligand adopts the preconformed structure in the absence of the receptor ( gpre ) , and a docking step , in which preconformed ligand and receptor associate ( gdock ) . the prime symbol indicates that receptor desolvation and nonelectrostatic contributions in the docking step , which are independent of ligand unbound conformation and ligand charge distribution , and , thus , are effectively constant , are neglected . each member of the collection of 1001 ligand conformations was treated individually as the unbound state , and the rigid - ligand charge optimization approach was used directly to optimize for chemically reasonable partial atomic charge distributions , leading to the most favorable binding free energy . the nonconvex optimization problem was solved using 100 multiple starts with conopt3 with the constraint qtotal = 0 ( neutral ligand ) . all trajectories reached the same solution , which indicates a wide basin of attraction for gtotal , in terms of the ligand partial atomic charges . figure 3a shows the computed optimal binding affinity gtotal for each of the 1001 individual unbound ligand conformations ( black symbols ) , as well as the preconformation ( gpre , blue symbols ) and docking ( gdock , red symbols ) contributions . dramatically different results were observed for rigid binding ( structure index i = 0 ; the unbound and bound ligand conformation are identical ) and nonrigid binding ( structure index i { 1 , 2 , ... , 1000 } ; the ligand changes conformation on binding ) . the binding affinity improved from roughly 20 kcal / mol for rigid binding to approximately 150 kcal / mol for nonrigid cases . kcal / mol ) and a modestly favorable docking change ( gdock 20 kcal / mol ) ; nonrigid binding involved a surprisingly favorable preconformational gain ( gpre of roughly 300 to 200 kcal / mol ) and an unusually large unfavorable docking change ( gdock of approximately + 100 to + 150 kcal / mol ; see figure 3a ) . that is , rigid binding charge optimization resulted in optimized charges that were truly complementary to the binding site ; flexible charge optimization resulted in charges that were not especially complementary to the binding site ( because gdock was substantially unfavorable ) but that pathologically recovered a large free - energy benefit from changing the conformation from unbound to preconformed free in solution . further analysis shows the favorable energetics result from a dramatically favorable coulombic gain ( gprecoul 500 kcal / mol ) and a more moderate desolvation loss ( gpresolv 200 kcal / mol ; see figure 4a ) . although the calculation assumes a given ligand conformation as the unbound state , optimization produces a charge distribution for which another conformation , namely , the preconformed structure , is lower in free energy . that is , the assumed unbound conformation is not the ground unbound state , because , conceptually , the system has a choice of two conformations ( p and u ) . we reasoned that elimination of this unphysical situation might lead to the charge optimization producing useful and meaningful results for flexible binding , which motivated the dominant - conformer unbound state method described in the next subsection . contributions to gtotal from three types of optimization : ( a ) the single - conformer unbound state method , ( b ) the dominant - conformer unbound state method , and ( c ) the dominant - conformer unbound state method , with the preconformed structure removed from the unbound candidate set . filled symbols are used for nl = 0 , whereas open symbols are used for all other values . are on the same scale . energetic and atomic - charge analysis of single - conformer studies : ( a ) contributions to gpre , ( b ) contributions to gdock , ( c ) root - mean - square deviation ( rmsd ) between single - conformer optimum and rigid optimum partial atomic charge distributions , and ( d ) rmsd between single - conformer optimum and nominal charge distributions . before presenting those results , we briefly comment on the mathematical source of the unphysical results . the ligand desolvation matrix ( the hessian of the electrostatic binding free energy ) for induced - fit charge optimization is l + lpsolv + lpcoul lusolv lucoul ( eq 22 ) , which is positive semidefinite for rigid binding ( where lpsolv = lusolv and lpcoul = lucoul and thus only l remains ) but need not be for single - conformation unbound state flexible binding . eigenvectors corresponding to negative eigenvalues present opportunities for improving ligand affinity without limit but are fixed by constraints placed on the ligand charge distribution . these directions produced outsized gains in gpre at the expense of gdock. indeed , examination of optimized charges indicates that many terminated at the constraint and differed substantially from both the rigid optimum charge distribution and the nominal one ( figures 4c and 4d ) . interestingly , the solvent - screened electrostatic contribution to gdock was favorable ( about 50 kcal / mol ; blue symbols in figure 4b ) but overpowered by the ligand desolvation penalty ( 100150 kcal / mol ; red symbols in figure 4b ) , which resulted in the unusual large unfavorable gdock. these mathematical sources of unphysical behavior correspond to explanations in terms of chemical interaction energetics . eigenanalysis revealed two mechanisms operating in tandem that were responsible for unphysical behavior . one mechanism was that atoms whose solvent exposure increased in going from the actual unbound to preconformed unbound state could improve electrostatic affinity by increasing charge magnitude on these atoms and gaining solvation interactions upon preconformation . the second was that atom pairs whose interatomic distance changed upon preconformation could gain affinity by accumulating like charge on atoms that became more distant ( or opposite charge on atoms that became closer ) upon preconformation . we next enforced the notion that the unbound state , if represented as a single conformation , should be the ground state among conformations represented in the set of unbound candidates . the new procedure involved a different formulation in which the optimization simultaneously chose a ligand partial atomic charge distribution and a presumed unbound ligand conformation from a set of candidates . the choice was made self - consistently , such that the presumed unbound ligand conformer did have the lowest free energy in the set when computed with the optimum charge distribution , and the optimum charge distribution was selected to minimize the total binding free energy change for preconformation and docking summed . if multiple unbound conformers had the same unbound state free energy ( to within 10 kcal / mol ) , they were considered equal contributors to the unbound state , each with the same gtotal. in a post - processing step , the energetic cost to gpre of using degenerate conformers for the unbound state was treated by adding an entropic penalty of rt ln ( is the number of energetically equivalent conformers ) . a series of individual optimizations was run using an unbound candidate set ( u ) of increasing size . i = 0 , the set was of the form u = { 0 , 1 , ... , nl}. in successive runs , nl was incremented from 0 to 1000 . the continuous and smooth formulation of the optimization in eq 25 was used . the computed optimal binding affinity gtotal was approximately 20 kcal / mol and did not vary systematically with nl ; this affinity was due to a zero preconformation energy gpre and dominated by gdock ( 20 kcal / mol ) . these results are dramatically different from those obtained with the single - conformer unbound state method ( see figure 3a ) . the new formulation incorporating the ground - state constraint both eliminated the large favorable gpre and maintained a favorable gdock. this latter point is consistent with intuition of electrostatic complementarity . the preconformation free - energy change was zero , which suggests the preconformed structure was selected either as the only unbound ligand conformation or as one of several . the coulombic , solvation , and nonelectrostatic contributions to the preconformation free energy were not always individually zero but summed to zero ( different from the single - conformer case ) , which suggests degenerate unbound conformers were sometimes selected . the docking free - energy contribution was similar to that for the rigid case . the charge distribution was closer to the rigid optimum than to the nominal distribution , but it could be distinct from either . thus , interestingly , much of the energetics of rigid binding was reproduced for this dominant - conformer case , although differences in energetics and charge distributions did result . the optimized ligand charge distribution qflex,2opt ( when nl = 1000 ) is reported in table 1 , along with that derived with a rigid ligand ( qrigidopt ) and the nominal charge distribution ( q ) . energetic and atomic - charge analysis of dominant - conformer studies : ( a ) contributions to gpre , ( b ) contributions to gdock , ( c ) rmsd between dominant - conformer optimum and rigid optimum partial atomic charge distributions , and ( d ) rmsd between dominant - conformer optimum and nominal charge distributions . ( a ) indices of selected unbound conformations for incrementally increasing unbound conformer candidate set for the dominant - conformer studies . ( b d ) some selected conformations of unbound mit-2-kb-98 aligned to each other . ( b ) conformer 44 ( cyan ) and its replacement conformer 953 ( gray ) ; conformer 0 ( black sticks ) was aligned to conformer 44 with all atoms . ( c ) conformer 907 ( cyan ) and 953 ( gray ) coexisted with conformer 0 as selected unbound conformers . ( d ) conformer 907 and its replacement conformer 996 only differed in the position of a hydrogen atom in the hydroxyl group attached to the benzene ring . q is the nominal charge distribution , qrigidopt is optimized using a rigid ligand , and qflex,2opt is optimized using a flexible ligand treated with the dominant - state unbound conformer approach using all 1001 candidates . figure 6a indicates the indices of conformations selected as the unbound state for each optimization . the preconformed structure ( index i = 0 ) was selected in every optimization , almost always with a small number ( up to 2 ) of other conformations of equivalent free energy . as the size of the unbound candidate set was progressively increased , certain structures were selected until replaced by newer ones . after nl reached 50 , conformer 44 was always chosen , along with conformer 0 , until replaced by conformer 953 . conformer 907 joined conformer 44 as one of the selected conformations until being replaced by conformer 996 . conformers 44 and 953 ( which replaced conformer 44 ) only differ in the orientation of the hydroxylated benzene ring ( see figure 6b ; upper right ) . in conformer 953 , it was almost in the same plane as the neighboring scaffold atoms n15 , c16 , n17 , and c4 ( labeled ) . conformer 907 resembled conformer 953 , except for a small torsional angle difference for the benzene ring and in the position of the hydroxyl group hydrogen atom ( figure 6c ) . these two conformers were selected , together with conformer 0 , until conformer 907 was replaced by conformer 996 , which only differs in the position of the same hydroxyl hydrogen atom . thus , the dominant - conformer unbound state method removed the pathological behavior observed in the single - conformer method through elimination of the unphysical situation of the unbound conformer not being the ground state structure . interestingly , this produced binding energetics extremely similar to rigid - binding charge optimization , and the preconformed ligand structure was consistently chosen as the unbound ground state , sometimes with one to two other degenerate structures . indeed , an analysis of the lagrange multipliers from the constrained optimization shows that even when degenerate conformers populated the unbound state , the cost for losing conformer 0 dominated the others substantially . the somewhat varying values for contributions to gpre are due to averaging different multiple members of the unbound ligand state for each optimization . taken together , these results suggest that there is little or no affinity benefit to changing conformation upon binding , and that adoption of unbound structures corresponding to complementary geometry in the bound state is sufficient to produce optimal affinity . this suggestion is strengthened by similar results obtained when constraining the total ligand charge to other values ( qtotal { 1 , + 1 } ) , as well as running similar jobs using a crystal structure rather than the design structure as the bound state and updating structure 0 accordingly ( data not shown ) . as one final control , we reran the dominant - conformer method with the preconformed structure ( index 0 ) removed from the unbound set , so u = { 1 , ... , nl}. this was done to isolate the role of the preconformed ligand structure in the unbound candidate set , because of the many methodological differences between the single - conformer and dominant - conformer methods . the overall results and detailed breakdown ( figure 3c ) were generally similar to the single - conformer case , with pathologically favorable affinity arising from unphysically favorable gpre values . these data imply that the preconformed ligand structure should always be considered as a candidate for the unbound conformation , because the alternative not only produces physically unrealistic results but also is physically untrue . here , we have extended charge optimization theory to treat potentially flexible ligands . one additional concept was used to ensure physically meaningful results any conformational change from the unbound state to the preconformed bound conformation must be uphill in free energy ( or at least neutral ) . if it were downhill , then the presumed unbound state would not correspond to the ground state . the addition of this constraint produces physically reasonable optima and eliminates pathologies that appear in its absence . the flexible binding case was extended from previous work on rigid binding , in which electrostatics could be clearly separated from nonelectrostatic contributions to the binding free energy . the theoretical treatment of the flexible case developed here intimately links nonelectrostatic with electrostatic contributions in the preconformational free - energy contribution in eq 22 . the conformational free - energy surface of the unbound ligand can , in principle , affect the optimization of the unbound conformation and its charge distribution . note that the parameters for calculating nonelectrostatic contributions are assumed independent of ligand partial atomic charges in the treatment presented here . in practice , these parameters are often calibrated to be compatible with these charges in force - field development , and so may be somewhat interdependent . moreover , to create a perturbed ligand with charge distribution closer to optimal , presumably one would need to alter covalent chemistry , which could further change molecular flexibility and the corresponding internal potential . nevertheless , an understanding of the charge optimum , in the presence of the current covalent potential , could be quite valuable . a feature of the problem formulation adopted here is that a set of candidate conformers ( including the preconformed ligand structure ) is presented as input to the optimization procedure . optimization selects a conformer ( or a few degenerate conformers ) for the unbound state and an optimized charge distribution that together minimize the total binding free energy while satisfying all constraints . as the set of unbound conformer candidates the direct effect is that there are more candidates from which to select the unbound conformation . the indirect effect is that if a new conformation is not selected as the unbound ground state , its presence is an additional constraint that limits the charge space available to the eventual ground state , because , to be the ground state , it must be lower in unbound free energy than all other conformers considered . for example , if the previous ground state is chosen as the ground state again , with the optimal charges applied it has to be lower in free energy when unbound than all other conformers including the new conformer added . for the test case used here , the preconformed ligand structure was always selected as the ( possibly degenerate ) ground unbound state by the optimization , even when the number of candidates grew to 1001 . for increasing numbers of candidates , the optimized charge distribution differed from the rigid optimum due to the indirect effect , which diminished the optimum binding affinity by over 0.5 kcal / mol for candidate sets with more than 20 members . for larger sets than those studied here , or for other cases , one imagines that a different conformation might be chosen for the ground unbound state , but this would be a result of needing to satisfy constraints rather than an adaptation to improve affinity . for the cases studied here , permitting flexibility never improved optimal affinity beyond that achieved by rigid binding optimization , and , frequently , the induced - fit case incurred an affinity penalty , relative to the rigid case . one interpretation of these results is that induced - fit binding , which may have a special role in molecular recognition , may not be an optimization to enhance affinity . this interpretation is based on the theoretical framework developed here , which includes partial atomic charge distributions that remain fixed upon molecular conformational change . it is formally possible that conformational change is designed to create appropriate charge distribution changes that do improve affinity , and we can not currently rule out this possibility . what we can say , however , is that conformational change upon binding does not appear to enhance affinity by improving the tradeoff between desolvation and interaction energetics , unless it also can somehow simultaneously lead to affinity - enhancing charge distribution changes . these arguments can be generalized beyond the specific molecular example studied here by noting that induced - fit binding can be conceptualized as a preconformation step , followed by a docking step , as illustrated by the blue row of figure 1 . constraining the selected unbound conformer to the ground unbound state means that the preconformation step has a free - energy change larger than or equal to zero , with a lower bound of zero . a lower bound on the docking step is the rigid - ligand electrostatic optimum , which is also a lower bound on gtotal. this lower bound will be an optimum solution for the induced - fit case unless it violates the ground - state constraint . thus , the dominant - conformer optimum can not have higher affinity than the rigid optimum , and any optimum producing an actual ligand conformational change upon binding will have affinity either equivalent to or weaker than the rigid optimization , and be a consequence of having to satisfy the ground - state constraint . the practical implication for molecular design of high - affinity ligands is that the construction of ligands preconformed for binding and with rigid - ligand optimized partial atomic charges leads to highest affinity in the theory developed here . if the ligand does still change conformation on binding , in many cases , the rigid - ligand optimized partial atomic charges will still be excellent . for the cases studied here , the largest loss in electrostatic affinity from this strategy would be 4.00 kcal / mol ( from 8.35 kcal / mol , which is calculated to be the optimal value ) , and the largest loss in total affinity would be 8.11 kcal / mol ( from 66.78 kcal / mol as the optimal value ) . the current work adds molecular flexibility to charge optimization theory but still treats each state as a single conformer . molecular systems actually exist as boltzmann - weighted distributions of conformations , and the current theory is being extended to treat such ensemble distributions , which could lead to new insights into the sources of conformational changes that accompany binding .

the design of ligands with high affinity and specificity remains a fundamental challenge in understanding molecular recognition and developing therapeutic interventions . charge optimization theory addresses this problem by determining ligand charge distributions that produce the most favorable electrostatic contribution to the binding free energy . the theory has been applied to the design of binding specificity as well . however , the formulations described only treat a rigid ligand one that does not change conformation upon binding . here , we extend the theory to treat induced - fit ligands for which the unbound ligand conformation may differ from the bound conformation . we develop a thermodynamic pathway analysis for binding contributions relevant to the theory , and we illustrate application of the theory using hiv-1 protease with our previously designed and validated subnanomolar inhibitor . direct application of rigid charge optimization approaches to nonrigid cases leads to very favorable intramolecular electrostatic interactions that are physically unreasonable , and analysis shows the ligand charge distribution massively stabilizes the preconformed ( bound ) conformation over the unbound . after analyzing this case , we provide a treatment for the induced - fit ligand charge optimization problem that produces physically realistic results . the key factor is introducing the constraint that the free energy of the unbound ligand conformation be lower or equal to that of the preconformed ligand structure , which corresponds to the notion that the unbound structure is the ground unbound state . results not only demonstrate the applicability of this methodology to discovering optimized charge distributions in an induced - fit model , but also provide some insights into the energetic consequences of ligand conformational change on binding . specifically , the results show that , from an electrostatic perspective , induced - fit binding is not an adaptation designed to enhance binding affinity ; at best , it can only achieve the same affinity as optimized rigid binding .

Introduction Theory Methods Results Discussion and Conclusion

PMC5241154

porous organic cages ( pocs ) are a unique class of microporous material composed of discrete molecules with intrinsic , guest accessible cavities ( figure 1a ) . to be porous in the solid state , these cavities must be connected by a 1- , 2- , or 3-dimensional pore network ( figure 1b ) . without this connectivity , the intrinsic cavities are isolated and inaccessible to guest molecules . the cages must also remain shape - persistent upon addition and removal of guests , such as solvent , because collapse of the intrinsic cavity would disrupt the pore network . the intrinsic porosity inside the cages may also be augmented by extrinsic voids between cages . the combination of these two requirements ( porous crystal packing and shape - persistence ) , coupled with the synthetic challenge of forming a cage in the first place , makes pocs easy to design on paper ( a ) space filling representation of the porous organic imine cage cc3-r , taken from its single crystal structure ( gray , carbon ; white , hydrogen ; blue , nitrogen ) . the cage is shape - persistent and has an intrinsic cavity that is accessible via four windows . ( b ) schematic representation of crystalline cc3 : each cage packs window - to - window with four adjacent cages to form a 3-d pore network . the intrinsic cage cavities are connected by extrinsic voids between the cage windows ( gray , core cage structure ; red , cyclohexane groups located on the cage vertices ; yellow , 3-d pore network ) . the cage packing has a dramatic effect on porosity , and different crystalline polymorphs of the same molecule can have quite different physical properties . pocs share some similarities with metal organic frameworks ( mofs ) , covalent organic frameworks ( cofs ) , and porous polymer networks , but because of their discrete molecular nature , they are usually solution processable . this processability allows pocs to be used in applications that would be more challenging with insoluble porous solids . to give one example , the molecular nature of pocs also gives options for characterization ( e.g. , solution nmr , hplc ) and purification ( e.g. , recrystallization , preparative chromatography ) that are unavailable for insoluble , extended frameworks . in the cooper group , we have primarily focused on the synthesis of imine pocs , but the following discussion is also relevant to cages formed using other bond forming reactions ( e.g. , boronate esters ) . our aim is to highlight more general experimental strategies by using cc1 and cc3 as detailed worked examples ( scheme 1 ) . there are a number of challenges involved in the design and synthesis of pocs ; this is especially true for completely new molecules , but even the preparation of structural variants of known pocs can present unforeseen difficulties . we have found that even subtle changes to bond angles in the precursors can have a dramatic effect on the outcome of the reaction by changing the size and stoichiometry of the cage product , as also found for metal organic cages . moreover , essentially any cage precursor combination can , in principle , form an amorphous polymer network instead of a cage . sometimes , a small change in one of the precursors or the use of unsuitable reaction conditions can flip the system from being cage - forming to being polymer - forming . high - dilution synthesis coupled with dynamic covalent chemistry is a common strategy to avoid this , but this will only succeed if the target cage is the thermodynamic product . the synthesis of imine cages from amine and aldehyde precursors is an example of this . dynamic covalent routes allow the thermodynamic cage product to emerge from the various kinetic products that are initially formed in the reaction , and this can lead in many cases to high synthetic yields ; for example , the yield of cc3 in batch syntheses usually exceeds 80% . we have synthesized up to 30 g of cc3 in a single batch ( figure 2a ) , and there is no reason to think that larger scale syntheses are not possible . ( a ) large scale batch synthesis ( > 10 g ) of cc3 yields a pure polycrystalline material . ( b ) large , millimeter - sized single crystals suitable for single crystal x - ray diffraction can also be isolated directly from reaction mixtures . obviously , it would be a major advantage to have methods to design appropriate precursors for poc synthesis without resorting to trial and error . in collaboration with the jelfs group , we are exploring methods to compute the size and shape of cages formed from a given set of starting materials , and their likelihood of remaining shape - persistent , before attempting any synthesis in the lab . for example , if the candidate cage structure is too strained , then it is unlikely to form . in time , we believe this will be a powerful and generalizable method for in silico poc design . for the moment , though , some intuitive design strategies exist . for example , if a cage is too flexible , then it will often undergo a structural rearrangement upon desolvation , resulting in collapse of the intrinsic cavity and loss of porosity . this can be due to the cumulative effect of small degrees of freedom in multiple rigid bonds as well as ( more obviously ) the inclusion of freely rotatable or highly flexible linkers . as a result , the design of large shape - persistent cages ( > 23 nm diameter ) is generally more difficult than for smaller pocs , and there are fewer examples of large cages in the literature . once potential cage precursors have been identified , then suitable synthesis , purification , and isolation conditions must be developed . parameters that can affect the outcome of the cage - forming reaction include concentration , temperature , and solvent and catalyst choice . whether or not water ( or other condensate ) is removed during the reaction can also be important as can the order and speed of the reactant addition . selection of the reaction parameters should be informed by the properties of the reactants , such as their reactivity and solubility . for instance , the wrong solvent choice can lead to premature precipitation of intermediates from the reaction mixture before the target cage is formed . once kinetic products precipitate , they may not be able to equilibrate into the desired cage , even if it is the thermodynamic product . the addition of solubilizing groups ( e.g. , alkyl chains ) is another strategy , although for pocs , this bears the potential disadvantage that these groups may diminish the porosity in the solid state or decrease the propensity of the cage to crystallize , if that is the goal . a different strategy to improve cage solubility is to decrease the symmetry in the cage , for example , by using mixed linkers , but this may strongly inhibit crystallization . it may also be necessary to add a catalyst , such as an acid , to enhance the reversibility of the dynamic covalent bond forming chemistry , although in some cases this can also direct the reaction toward other products , such as interlocked catenanes . with sensible precursor selection and optimization of the reaction conditions , also , if there is sufficient preorganization in the cage precursors , dynamic covalent chemistry may not always be required . with luck , the cage may crystallize directly from the reaction mixture in a porous phase that remains stable to direct desolvation ( e.g. , cc3 ) , but this is relatively uncommon . even if a cage does crystallize directly from the reaction mixture , it may not be easy to determine whether insoluble oligomers are also present as side products , especially if the cage itself is poorly soluble . in addition , amorphous cage or side products might not be revealed by powder x - ray diffraction ( pxrd ) ; we have found that a significant proportion of amorphous material can be present before there is a noticeable change in the baseline of the pxrd . hence , even cages that appear to be chemically pure and phase pure ( e.g. , by solution nmr and by pxrd ) might still be contaminated , for example , with a small quantity of insoluble amorphous polymer that is invisible to these techniques . moreover , crystals that grow directly and in some cases rapidly from a reaction mixture might be of lower quality and possess a greater number of defects than crystals grown in a more controlled process . this is an important consideration because crystal quality can strongly affect porosity . quite often , the desired cage product can remain in solution , perhaps with some insoluble polymeric byproduct . in such cases , one should take care not to mistake this precipitate for the cage product and discard the supernatant ! indeed , even if the cage product crystallizes from the reaction solvent , the supernatant may contain a significant quantity of cage , which can be recovered . a further complication , often overlooked , is that even a 100% yield of the cage does not automatically mean that it is the thermodynamic product . provided that there are no steric clashes , the global thermodynamic product may instead be a catenated cage where two ( or conceivably more ) independent cages become mechanically interlocked . in such cases , however , because catenation often requires extended reaction times and conditions that promote reversibility , such as higher temperatures or the use of a catalyst , then the noncatenated cage can often be isolated even if it is not the overall thermodynamic product . more generally , minor side - products , whether catenanes or other species , need not be fatal to success : as discussed in the methods section , various chromatographic , precipitation , and crystallization methods can be used to isolate cages as chemically pure single components . finally , solvent choice can play a part in determining the size and shape of the cage . for example , warmuth et al . demonstrated that three different cages could be formed from the same starting materials simply by changing the reaction solvent . recently , we also found that other imine cages can equilibrate to form new cage products on prolonged standing in certain crystallization solvents , suggesting that a kinetic cage product is formed initially . once pure cage material has been isolated , we would typically screen a range of conditions to afford either crystalline or amorphous phases of the cage , as required . usually , crystalline phases will be isolated as solvates , although in rare instances the crystallization solvent may be excluded from the crystal , particularly if the cage cavity is small and the solvent is large . crystals may also spontaneously and rapidly lose solvent at ambient temperature in air while remaining crystalline , particularly if the solvent is volatile and weakly interacting ( see figure s19 in ref ( 1 ) ) : in such cases , it might appear that the cage is crystallizing solvent free unless the material is characterized immediately upon isolation from the crystallization solvent . more commonly , the crystalline solvated phase must be carefully desolvated , or activated , to isolate a porous solid . as observed for mofs and cofs , activation is generally more challenging for pocs with low density ( high pore volumes ) and for solvates where the solvent has a high boiling point , particularly if the solvent strongly interacts with the poc framework , for example , by hydrogen bonding . in such cases , solvent exchange for a less polar , more volatile solvent prior to solvent removal may be necessary . pxrd in combination with electron microscopy can be used to establish whether the cage has changed phase or become amorphous after desolvation . also , if gas adsorption is to be performed , we strongly advocate repeating these characterization techniques after the adsorption measurement to ensure that the cage has not changed phase . not all applications require crystallinity ; for some purposes , amorphous pocs may be advantageous . techniques to isolate amorphous phases , or defective crystalline materials , include chemical scrambling , freeze - drying , rapid precipitation , spin - coating , electrospray , and ( conceivably ) mechanical grinding . throughout each stage of synthesis , isolation , and properties evaluation , analytical methods should be used to ensure that the purity and structure of the cage material is unchanged . for example , hplc and solution nmr are simple methods for ensuring chemical stability , and pxrd and electron microscopy , as discussed above , can be used for phase identification at any stage in the process . all of these methods require only small ( < 20 mg ) quantities of material . our most studied pocs , cc1 and cc3 , are synthesized by a [ 4 + 6 ] cycloimination reaction in which four molecules of the aldehyde 1,3,5-triformylbenzene ( tfb ) react with six molecules of either ethylenediamine ( eda , cc1 ) or 1r,2r-1,2-diaminocyclohexane ( chda , cc3-r ) ( scheme 1 ) . to date , we have published two separate high yielding procedures for the synthesis of cc1 and cc3 : a batch synthesis that affords multigram quantities of poorly crystalline cc1 or crystalline cc3 within a week and a continuous flow synthesis that affords milligram quantities of amorphous cc1 and semicrystalline cc3 within minutes and gram quantities within hours . in the batch synthesis of cc3 , a solution of homochiral trans - chda in dcm is layered onto a suspension of tfb in dcm containing a catalytic amount of tfa . the concentration is relatively high compared with standard cage or macrocycle reactions , which avoids excessive solvent volumes . however , the slow dissolution of the poorly soluble tfb over a number of days , along with the crystallization of cc3 from the supersaturated reaction mixture , is essentially equivalent to running the reaction at high dilution with the slow addition of the tfb . similar approaches that use solubility tuning of the reagents should be possible with other cages . the rigidity and homochirality of chda ensure that the intermediates are at least partially preconfigured to form the cc3 cage , thus reducing the formation of misaligned kinetic oligomers in comparison with more flexible reactants . indeed , in this system , no oligomeric side - products are observed to precipitate from the concentrated reaction mixture . the role of the acid catalyst is to increase the reversibility of the imine reaction and to afford the thermodynamic cage product within a reasonable time frame . the use of a catalyst is particularly suited to the synthesis of cages that use less reactive starting materials . heating may also be employed either to increase reversibility or in cases where the reactants or the cage product has poor solubility . the isostructural poc , cc1 , formed by the batch reaction of tfb with eda ( scheme 1 ) , is synthesized under different conditions . this reaction is run at low temperature and high dilution with slow addition of tfb to the diamine . these conditions are required to mitigate the higher reactivity and inherent flexibility of this diamine , which can result in the formation of insoluble oligomeric products at higher concentrations and higher reaction temperatures . hence , this route is more reminiscent of classical macrocycle syntheses . to synthesize cc1 , a solution of tfb in dcm is added dropwise over 48 h to a solution of the eda in dcm at 0 c . the use of a syringe or peristaltic pump rather than an addition funnel ensures more accurate control of addition rates and better reproducibility . after warming the reaction mixture to room temperature and stirring for a further 24 h , the starting materials show complete conversion to cc1 with no soluble byproducts observed . the high reactivity of the starting materials means that an acid catalyst is not required even at high dilution . in fact , when the reaction is run in acetonitrile , the presence of an acid catalyst affords catenated cc1 , which is the true thermodynamic product.cc3 , on the other hand , is unable to catenate due to the presence of the bulky cyclohexane groups on the cage vertices . catenated cages are easily spotted because they often display a marked change in their h nmr spectra due to a decrease in symmetry and a pronounced through - space anisotropic effect caused by close contact between the interlocked cages . they can also be identified , of course , by techniques such as mass spectrometry , hplc , or diffusion - ordered nmr spectroscopy ( dosy ) . the formation of cc1 and cc3 does not appear to be sensitive to the water liberated during the reaction , either in batch or continuous flow conditions . however , we have encountered other cages that require the removal of water via a dean stark apparatus ( a reverse dean stark apparatus can be used for chlorinated solvents ) or the addition of a desiccant to ensure good conversion of the reactants to the cage product . this requirement is often associated with the low reactivity of one of the starting materials due to steric or electronic effects . as a rule of thumb for the synthesis of imine cages under batch reactions , reactive starting materials require high dilution ( unless solubility is controlled ) , low temperatures , and slow addition of one of the precursors ( again , unless solubility can act as a control ) . less reactive starting materials tend to require the use of an acid catalyst , higher temperatures , more concentrated reaction mixtures , and the removal of water ( e.g. , with a dean stark trap ) to improve conversion . generally reactions proceed more cleanly when the aldehyde is added to an excess of diamine ; the excess amine in the reaction helps to promote reversibility and should minimize oligomer formation . the synthesis of cc1 and cc3 has also been successfully transferred to a vaportec r - series continuous flow reactor as a proof of concept . by using flow systems , the reaction temperature can be increased above the boiling point of the solvent . coupled with the highly efficient reagent mixing characteristic of flow systems , this allows the rate of reaction to be increased and the reaction time to be reduced , in some cases from days to minutes . the short reaction time allows multiple combinations of starting materials to be assessed rapidly across a range of conditions . concentration , stoichiometry , reaction time , flow rate , and temperature are all easily varied . the synthesis of cc3 under flow conditions afforded full conversion of tfb to cage in just 10 min at 100 c , whereas the batch synthesis takes a number of days . a stoichiometry of 4 tfbs to 6.5 diamines was used in the reaction ( ideal stoichiometry is 4:6 ) to mitigate fluctuations in the pump performance . the excess diamine was well tolerated and ensured complete conversion of tfb to the cage , whereas the use of excess aldehyde resulted in incomplete cage formation . the same effect has been noted before in our group in the batch synthesis of imine cages , where the use of a small excess of amine can result in cleaner and more reproducible reactions . the effect of temperature and reaction time on the flow synthesis was also studied with lower temperatures or shorter reaction times affording incomplete conversion to the cage product . higher temperatures resulted in the precipitation of oligomeric material , whereas a longer reaction time had no effect on the outcome of the reaction . surprisingly , we also found that cc1 could be synthesized using the same reaction conditions despite significant differences between the batch synthesis conditions for each cage ( above ) , and the reduced thermal and hydrolytic stability of cc1 . unfortunately , due to the poor solubility of tfb in dcm , we were unable to test the effect of more concentrated reaction mixtures on the outcome of either reaction using this flow system . however , the flow synthesis of cc1 is still three times more concentrated than its batch synthesis , significantly reducing solvent volumes . for both cc1 and cc3 , the reaction stream exiting the reactor afforded a solution of the cage contaminated with only the excess diamines , which are easily removed by antisolvent reprecipitation . we have found several examples of cages that form in solution in good yields but are sensitive to the isolation procedure and can readily decompose into an insoluble polymeric material upon solvent removal . one simple monitoring technique is to dilute a sample of the reaction mixture with a suitable deuterated solvent for analysis by h nmr . although the signals of interest may be weak and partially obscured by the nondeuterated reaction solvent , such spectra often provide sufficient information to determine whether the aldehyde has been consumed and how cleanly the reaction has progressed . if the target cage is symmetrical , then the h nmr spectrum is usually relatively simple . a more complex h nmr would potentially indicate the formation of oligomeric products , incomplete conversion to the cage , or potentially catenated products . ms , which can give information on the purity and composition of the reaction mixture . a final technique that can be used directly on reaction mixtures is dosy . to do this , however , the reaction should be performed in deuterated solvents . with careful calibration , dosy can be used to determine the size and hence stoichiometry of the cage that is formed , if any . for reaction mixtures that form a suspension ( e.g. , batch synthesis of cc3 ) , it is good practice to analyze a sample of both the solid and the supernatant to determine what quantity , if any , of cage has formed and its purity in each phase . depending on the composition of the solid and the supernatant , it is often easier to redissolve the solid and combine it with the supernatant to purify the material as a single batch . obviously , if the quantity of cage in the supernatant ( or in the precipitated solid ) is insignificant , then that phase can be discarded . if the cage is mainly present in the solid phase , and even if it gives a pxrd pattern consistent with cage formation , this does not guarantee that it has crystallized phase pure : it is still good practice to dissolve some of this material and to check that it is not contaminated with oligomeric materials or other side products . pocs are organic molecules , and they can be characterized by the standard range of organic chemistry techniques . also , as discussed above , crystallization of the cage from the reaction solvent may not produce good crystals , although in several cases we have obtained excellent quality millimeter - sized crystals directly from reaction mixtures ( figure 2b ) that were suitable for single crystal x - ray diffraction . in many cases , though , it is advisible to grow crystals of purified material in a subsequent step , although this may produce a different polymorph to that which precipitated from the reaction mixture , especially if a different crystallization solvent is used . in this respect , soluble pocs differ significantly from insoluble mofs and cofs where the crystals must , by necessity , be used as synthesized . the first priority when developing an isolation procedure to yield a porous crystal ( or an amorphous poc solid ) is to check that you are starting with chemically pure material . if the chemical purity of the cage is poor , then it can make the subsequent isolation of crystalline phases more difficult . in the batch synthesis of cc3 , the cage crystallizes directly from the reaction mixture in good yield and excellent chemical purity , though this is not always the case for all pocs . pure cc3 is isolated by filtration of the reaction mixture , followed by a displacement wash with 95:5 etoh / dcm to remove any surface impurities . cc1 , on the other hand , remains in solution when the reaction is complete . the reaction mixture is first filtered to remove any traces of polymeric material that have formed during the reaction , and this filtered solution is evaporated to dryness at < 20 c ; higher solvent evaporation temperatures can result in polymerization of the cage . indeed , because of the extended evaporation time as a result of the large reaction volume , some cage polymerization can still occur , even at low temperatures . hence , after reaching dryness , the residue is redissolved in a small volume of volatile solvent , usually dcm , refiltered to remove any insoluble polymeric material , and this much smaller volume of solvent evaporated at < 20 c to afford cc1 as a chemically pure solid . more generally , dissolution and filtration is a good method to remove any contamination with insoluble oligomeric side - products . in the flow synthesis of cc1 and cc3 , both products exit the reactor as a dcm solution that also contains excess diamine . cc3 is precipitated directly from the reaction mixture by mixing it with excess hexane . however , because of the higher solubility of cc1 , the reaction mixture must first be evaporated ; the material is then redissolved in a small amount of solvent , filtered to remove any polymeric material formed during the evaporation , and precipitated by addition to hexane . in both cases , the product is then isolated by filtration to yield the cage in excellent purity and yield a number of factors affect the choice of antisolvent : it must be miscible with the cage solution and induce precipitation of the cage in good yield and purity . comparing the hplc of the initial cage solution with the supernatant of the precipitated suspension allows rapid screening of solvent / antisolvent combinations ; in a successful purification , the filtrate will be heavily enriched in impurities as the cage will have precipitated . a related technique that we often use to isolate cages is to swap the solvent , often dcm or chloroform , to a higher boiling antisolvent . the cage solution is usually diluted with the antisolvent , which may result in a cloudy solution / suspension , and then the original solvent is slowly evaporated to leave a suspension of the cage . the cage can then be isolated by filtration while more soluble impurities ( e.g. , unreacted monomers ) remain in solution . alkanes , such as hexane , and acetone have proven to be particularly effective antisolvents for this technique . we routinely employ this technique to isolate soluble cages directly from their reaction mixtures , and in some instances , it has led directly to the formation of a crystalline porous phase . , common crystallization solvents can be screened on a small scale with heating to establish the solubility of the cage . on cooling , the recovery and purity of any cage precipitate can again be assessed by hplc analysis of the supernatant . for recovery to be increased , an antisolvent may be added to the hot cage solution . cages that are thermally unstable may also be purified by adopting the ambient temperature crystallization techniques outlined below . the correct solvent choice for any of these methods is based on a combination of experience and ( substantial ) trial and error . if pure cage can not be obtained using any of the methods discussed , then chromatography may be used for purification . we have had the most success with preparative hplc and have been able to separate desired cage products from soluble impurities , usually cage fragments or oligomers , isostructural cages , and catenated cages . in our hands , purification usually involves injecting a solution of the crude cage onto a c8 reverse phase column then eluting with methanol , the presence of imine bonds in the cages precludes the use of water - containing gradients . unsurprisingly , we have found huge differences in performance between column manufacturers ; hence , the suitability of a column should first be assessed using an analytical system . other chromatographic techniques , such as size exclusion chromatography , have also been investigated , but these have so far met with limited success in our experience . the purification of pocs by vacuum sublimation , the organic chemist s last resort , is a further possibility , although the vapor pressure of these relatively large macrocyclic molecules may often be too low for this to be viable . unlike insoluble mofs and cofs , the porosity in pocs can be modified by postsynthetic crystallization steps . as such , isolation of a cage from a reaction mixture that is initially found to be nonporous cc3 crystallizes from the batch reaction mixture in a single desolvatable phase , cc3 , where the intrinsic cage cavities are connected via extrinsic cavities to afford a 3-dimensional pore network ( figure 1b ) . however , as mentioned above , the porosity is influenced by the quality of the crystals that are formed . we have found that the apparent brunauer emmett teller surface area ( sabet ) of highly crystalline cc3 is , reproducibly , 409 8 m g , whereas standard as - made cc3 has been reported with sabet values of 592 and 624 m g. this is thought to be due to defects , such as missing cages , in the crystal packing , which add to the porosity in the material . the only discernible difference between the pxrd patterns of the highly crystalline and as made a polymorph screen with cc3 identified a second crystalline phase , cc3 , which crystallizes from dcm / diethyl ether and has an sabet of 555 m g when desolvated . hence , different levels of porosity can be obtained for the cc3 molecule depending on the method of crystallization . deliberate amorphization of cc3 via freeze - drying of cc3 from a solution of dcm affords material with an increased sabet of 859 63 m g. this increase in sabet upon amorphization observed for cc3 is not a general phenomenon ; more commonly , amorphization results in a decrease or a complete loss of porosity , as found for the isostructural cage cc1.cc3 increases its porosity in the amorphous state because the bulky cyclohexane groups on the cage vertices prevent efficient packing ( cc1 lacks these bulky groups ) . cc1 is isolated from the batch and flow reaction mixtures as a poorly crystalline solid , but a screen of different reaction and crystallization solvents has led to the discovery of a number of crystalline solvates . cc1 , cc1 , cc1 , and cc1 were isolated from ethyl acetate , dcm , o - xylene , and dioxane , respectively . upon desolvation , cc1 and cc1 undergo subtle structural changes to yield the related desolvated phases , cc1 and cc1. each polymorph was observed to possess different porous properties , including different gas selectivities . cc1 and cc1 are formally nonporous to nitrogen , whereas cc1 , when desolvated , has an sabet of 550 m g. cc1 was found to be unstable to desolvation and afforded a mixture of unidentified phases . interestingly , it was also found that nonporous and porous phases of cc1 could be interconverted in the solid state simply via exposure to solvent vapor . the first is to identify new crystalline phases , initially as solvates , and to obtain crystals of sufficient quality to allow them to be solved by scxrd . once crystalline phases have been identified , the next step is to isolate sufficient phase - pure material to allow characterization of physical properties . this can be more difficult than isolating the chemically pure cage in the first place . bulk samples should be analyzed by pxrd to ensure that they are phase pure and that they match the simulated pxrd from the solvated scxrd . electron microscopy can also be used to ensure sample homogeneity as secondary crystalline or amorphous phases should be visible . we routinely use a number of techniques to screen for crystalline phases of organic cages . single or polycrystalline phases can be obtained by slow evaporation of single or mixed solvent cage solutions under a nitrogen flow ; this may be achieved using a desiccator with a gas inlet on the lid . the solutions should be checked regularly for the appearance of suitable crystals for scxrd during the evaporation . after solvent evaporation , the bulk material should also be analyzed by pxrd : a common pitfall here is to select a single crystal that is , in fact , not representative of the bulk solid . vial - in - vial diffusion of an antisolvent into the cage solution or slow diffusion of an antisolvent layered onto the cage solution can also yield single crystal or polycrystalline materials . these materials will most likely be isolated as solvates unless the material spontaneously undergoes desolvation upon removal from the solvent . rapid desolvation of single crystals upon removal from the crystallization solvent may also be accompanied by a loss of singularity , although the material may still be polycrystalline . in practice , it is often better to keep the crystal in the crystallization solvent until it can be analyzed by scxrd . particularly sensitive crystals may be stabilized by encapsulation in a protective oil before analysis . one should also be wary of predicting properties from the solvated scxrd structures unless the material can be desolvated without a significant change in crystal packing . because pocs can be recrystallized after synthesis , it is also possible to produce cocrystals in a modular way containing two or more chemically distinct cages . clearly , the main technique for proving such structures is single crystal x - ray diffraction . however , because it is possible to form cocrystals with significant positional disorder , one may also need to exploit the solubility of pocs , for example , by using hplc and/or nmr to prove that the chemical composition of the cocrystal is the same as that suggested by the x - ray structure refinement . this is another key difference between pocs and mofs , cofs , and porous organic polymers : their chemical composition can be determined accurately using solution phase analyses without resorting to techniques such as acid digestion or other chemical decomposition . removal of solvent from poc solvates can have a number of effects . in an ideal case , the solvent can be removed with no or little change in the crystal packing , such that the pxrd of the activated material matches the pattern calculated from the solvated single crystal structure . sometimes , desolvation leads to a phase change where a new crystalline phase ( or a mixed phase ) forms . alternatively , and particularly with large and/or flexible cages or highly solvated structures , desolvation can lead to complete amorphization . lastly , and less widely recognized , desolvation can sometimes be accompanied by chemical decomposition of some or all of the cage material . various activation methods for porous materials have been used by our group and by others , including vacuum drying , supercritical drying , and solvent exchange . thermogravimetric analysis of the solvated material is a useful guide to the desolvation temperature required while also providing information on the thermal stability of the cage . the best method of activation is dictated by the stability of the crystal packing in the solvate , the boiling point of the crystallization solvent , and the strength of the interaction between the solvent and the cage . the diamondoid window - to - window packing observed for cc3 is a particularly stable self - reinforcing packing motif , and cc3 survives rapid desolvation at 80 c in a vacuum oven with no phase change or loss of crystallinity . however , not all cages , or indeed polymorphs of the same cage , possess such a strong packing motif , and abrupt desolvation can lead to mixed phases or partial amorphization . such materials will have different properties to their phase pure constituents , and their gas sorption isotherms may not agree , for example , with isotherms predicted from idealized scxrd structures . careful solvent exchange , via one or more solvents , to a lower boiling weakly interacting solvent is the most successful method so far for gentler desolvation of pocs , but it is likely that techniques such as supercritical drying , as used successfully for mofs , should also be applicable . ideally , one should also try and obtain a desolvated single crystal structure to provide an accurate structure of the activated poc . in practice , this can be difficult to achieve , particularly if the crystal structure is heavily solvated . solvent removal can lead to cracking of the crystals , and single - crystal - to - single - crystal transformations are substantially rarer than crystal - to - crystal transformations . quite often , though , the solvent can be removed while the crystal is still mounted in the diffractometer by slowly heating under a flow of nitrogen . cc3 , for example , can be readily desolvated in situ by heating to 117 c . pocs should be analyzed postactivation by h nmr to ensure that no chemical decomposition has occurred and also that the material has been fully desolvated : here , again , there is an analytical advantage over mofs and cofs in that the whole sample can be dissolved and any entrained guests released without chemically decomposing the poc . it should be noted that pocs often readily adsorb atmospheric moisture , which could affect any porosity measurements . this can also affect elemental analyses significantly : a poc can adsorb several weight percent of water , depending on ambient humidity , which is not an issue typically encountered with the elemental analysis of dense , nonporous organic compounds . therefore pxrd and , ideally , electron microscopy analysis should also be used to ensure that no phase change or amorphization of the sample has occurred when removing the solvent . these tests should be repeated after gas sorption measurements or exposure to other guests , such as solvent vapors , to ensure that the poc is still chemically and phase pure . finally , all analysis and characterization should be carried out on a single batch of material to ensure that the measured physical properties match those predicted from the poc structure . throughout the poc discovery process , a number of analytical tests are required to ensure that chemical and phase purity are maintained . again , pocs are molecules , and it is possible to analyze more than simple phase purity by pxrd , which should be the minimal analysis requirement . without these various analytical tests , it may be impossible to correlate physical properties with structure : for example , one may end up trying to correlate a gas sorption isotherm calculated from a single crystal x - ray structure with a physical measurement derived from a sample that has become partially amorphous , changed phase , or chemically decomposed . figure 3 provides an overview of our standard cage discovery workflow and the minimum analytical requirements recommended for each stage of the discovery process . overview of the cage discovery process and the chemical and physical analysis needed at each stage . we hope that this perspective raises awareness of the pitfalls that may be encountered along the road to discovering new pocs while at the same time suggesting solutions to these problems . the field of pocs is wide open for development , but progress requires a combination of techniques developed by organic and supramolecular chemists , coupled with methods borrowed from the world of extended framework solids . in this respect , it is a mistake to think of this area as organic chemistry or as materials chemistry : it is both . problems occur when either the synthetic chemistry ( e.g. , insufficient purification ) or the physical chemistry ( e.g. , wrong activation conditions ) are neglected . however , by approaching pocs in a systematic way , it is possible to discover truly remarkable materials . for example , there are now organic cage molecules in the literature with surface areas exceeding 3,500 m g that begin to rival the most porous mofs and cofs . dcm ( 100 ml ) was added slowly onto solid 1,3,5-triformylbenzene ( 5.00 g , 30.9 mmol ) without stirring at room temperature . trifluoroacetic acid ( 100 l ) was added directly to this suspension as a catalyst for imine bond formation . finally , a solution of 1r,2r-1,2-diaminocyclohexane ( 5.00 g , 44.6 mmol ) in dcm ( 100 ml ) was layered onto the suspension . the unmixed reaction was covered and left to stand at ambient temperature . over 5 days , all of the solid 1,3,5-triformylbenzene was consumed , and octahedral crystals of cc3-r grew on the sides of the vessel . the white , crystalline product was removed by filtration and washed with 95:5 etoh / dcm ( 25 ml ) . the solid was dried to constant weight at 80 c in a vacuum oven , resulting in a yield of 6.5 g ( 83% ) . the vaportec reactor was assembled using the r-2+/r-2 pump modules with the r-4 reactor module . once the system had reached steady state , the suspension was collected for 110 min . the suspension was isolated by filtration and dried to constant weight in a vacuum oven at 60 c to afford cc3-r as a white crystalline powder ( 0.918 g , 95% ) . schematic of flow reactor setup showing the optimized parameters for the synthesis of cc3-r . flow system setup : system solvent , dcm ; reagent a , 0.083 m 1r,2r-1,2-diaminocyclohexane in dcm ( 0.948 g/100 ml of dcm ) ; reagent b , 0.083 m 1,3,5-triformylbenzene in dcm ( 1.34 g/100 ml of dcm ) ; reagent c , hexane ; flow rate a , 0.62 ml / min ; flow rate b , 0.38 ml / min ; flow rate c , 4 ml / min ; reactor volume , 10 ml ; reactor temperature , 100 c ; back pressure regulator , 8 bar . a solution of 1,3,5 triformylbenzene ( 3.75 g , 23.1 mmol ) in dcm ( 1150 ml ) was added dropwise over 48 h ( 0.3 ml / min ) via pressure equalizing dropping funnel ( or a syringe or peristaltic pump ) to a solution of ethylenediamine ( 2.08 g , 34.7 mmol ) in dcm ( 850 ml ) at 0 c . after complete addition , the reaction was allowed to stir for another 24 h at room temperature . the solvent was removed from the filtrate via rotary evaporation ( temperature of the water bath maintained below 20 c ) ; the crude product was redissolved in chcl3 ( 100 ml ) , and the solution was refiltered . the residue was washed with chcl3 ( 50 ml ) , and the combined organic filtrate was concentrated under vacuum on a rotary evaporator ( temperature of the water bath maintained below 20 c ) to give the product as a beige powder with a yield of 4.25 g ( 94% ) . the vaportec reactor was assembled using the r-2 + pump module with the r-4 reactor module . once the system had reached steady state , the reaction mixture was collected for 53 min . the solution was evaporated to dryness at 20 c , and the residue was redissolved in the minimum amount of dcm , filtered , and poured into an approximately equal volume of hexane to afford a white suspension . the solid was collected by filtration , then dried to constant weight in a vacuum oven at 50 c to afford cc1 as a beige powder with a yield of 0.310 g ( 93% ) . schematic of flow reactor setup showing the parameters for the synthesis of cc1 . flow system setup : system solvent : dcm ; reagent a , 0.083 m ethylenediamine in 1:3 meoh / dcm ( 0.499 g/(25 ml of meoh + 75 ml of dcm ) ) ; reagent b , 0.083 m 1,3,5-triformylbenzene in dcm ( 1.34 g/100 ml of dcm ) ; flow rate a , 0.62 ml / min ; flow rate b , 0.38 ml / min ; reactor volume , 10 ml ; reactor temperature , 100 c ; back pressure regulator , 8 bar .

porous organic cages present many opportunities in functional materials chemistry , but the synthetic challenges for these molecular solids are somewhat different from those faced in the areas of metal organic frameworks , covalent organic frameworks , or porous polymer networks . here , we highlight the practical methods that we have developed for the design , synthesis , and characterization of imine porous organic cages using cc1 and cc3 as examples . the key points are transferable to other cages , and this perspective should serve as a practical guide to researchers who are new to this field .

Introduction Design and Synthesis of New POCs POC Isolation and Characterization Methods Summary Experimental Section

PMC2801005

design of new medical drugs is an exhaustive process with many challenges . that is , they are used to examine different compounds with a desired activity and extract some common substructures that provide this activity . several methods are proposed to determine a mathematical equation correlating different properties of the compounds . these methods are called as quantitative structure activity relationships methods . several other methods known as qualitative - sar methods examine different compounds and extract some common significant substructures with desired activity . in this paper , the main focus is a qualitative - sar method that uses active fragments as a map to guide for molecular design . an active fragment ( pharmacophore ) is a set of similar structural features in the structure of active molecules ( or drugs ) that is responsible for their biological activity . a graph is a kind of data structure that consists of a set of nodes and a set of edges between them . for example , in a 3d fully weighted molecular graph representation , each node may represent characteristic features of atoms ( e.g. , electrical properties ) , and each edge may represent characteristic features of bonds between them ( e.g. , length , value of wiberg 's index , and so on ) . therefore , researchers study frequent subgraph mining approaches [ 2 , 3 ] to find frequency of common fragments ( e.g. , pharmacophores ) , which is a collection of bonds and atoms in the structure of compounds with the same activity for a specific disease . the success of the proposed methods relies on the features used for the representation . as the complexity of representation increases , interpretation of the represented molecular information becomes harder , because the graph - based approaches usually have high complexities . for example , while comparing two molecules in terms of their structure and activity , it may be necessary to compare their substructures , which are represented as graphs . unfortunately , deciding whether two graphs have identical topological structures or not has an unknown complexity . the subproblem that is deciding whether one graph is a subgraph of another or not is known to be np - complete . therefore , frequent subgraph mining approaches may not find exact solutions in a bounded time because of their high complexities . researchers use heuristics to reduce search space while solving subgraph mining problems [ 5 , 6 ] . in this way , they try to get rid of high computational burden inherent to these problems . for example , subdue algorithm uses a greedy search technique for frequent subgraph mining to reduce the time complexity with a possible cost of missing some important substructures . as stated above , many researches convert the problem of discovering frequent substructures in active molecules into a frequent subgraph discovery problem , in order to use graph mining approaches . however , current graph mining approaches can determine frequent subgraphs only if these subgraphs repeat exactly the same in a graph database . therefore , in many settings , these methods may not find the most informative substructures that do not exactly repeat in the molecules but exist in those molecules with some fine differences . furthermore , because these approaches work like black boxes , domain experts can not easily interrupt the process to incorporate their knowledge into the solution . that is , another problem related to current substructure discovery approaches is their blind calculations . therefore , even if these approaches find solutions , their solutions should be extensively examined , tested , and verified at the end by the domain experts . this leads to a system where any failure in the early stages of the process can not be corrected until the overall process is completed . alternatively , in this paper , we advocate directly incorporating domain knowledge into the solution process at different levels . for this purpose , we propose to use a data pipelining approach , where domain experts can intervene the overall process to enhance the solution with their knowledge and expertise . more specifically , in this paper , we propose a visual data mining method for frequent substructure extraction , where histogram - based filtering method is used to reduce the search space . in our approach , graph representations of molecules are projected into a 3d feature space ( named atom - bond - atom space ) . each bond of a molecule ( an edge with nodes at each end in the graph ) is represented as a point in this space . when we project all of the molecules with a certain activity into this space , the resulting points compose clusters of bonds that are repeated in the structure of the active molecules ( possibly with fine differences ) . however , discovering these clusters is nontrivial because of the noisy points that represent infrequent bonds . at this stage , we filter the noise using a histogram - based visual data mining method so that the clusters can be discovered more clearly by various clustering algorithms . once the clusters are discovered , frequent substructures of active molecules are computed . during all these steps , a domain expert can intervene to guide the system if necessary ( e.g. , during filtering , clustering , and so on ) . we demonstrate how our approach can determine frequent substructures of molecules step by step through a case study , using the tuberculosis dataset from literature [ 7 , 8 ] . our experiments show that our approach can successfully determine active fragments that account for the activity of those molecules . we lastly show how the determined fragments can be used as features to automatically categorize new molecules as active or inactive with respect to a specific disease . we experimentally evaluate the performance of the proposed approach with respect to other approaches from literature . there are two groups of sar methods . the first one is known as quantitative - sar , which derives a correlation between the descriptors of molecules and their activity . then , different machine learning techniques are applied to the prepared dataset as described in literature to learn how to classify molecules . linear discriminant analysis ( lda ) , multilayer perceptrons , support vector machines ( svm ) , k - nearest neighbors ( k - nn ) , simulated annealing , partial least squares , linear regression , and classification trees are the most studied methods on quantitative - sar for classification of molecules [ 9 , 10 ] . qualitative - sar is the second group of approaches which is interested in finding some common substructures in the structure of molecules . there are many approaches related to qualitative - sar in literature which are based on frequent substructure mining methods . some qualitative - sar approaches use graph mining methods to find common substructures that exist in active molecules with high probability , but exist in inactive molecules with low probability [ 2 , 3 ] . our research is related mainly to two important research areas : frequent substructure extraction and visual data mining . especially in qualitative - sar applications , graph - based data mining approaches are used to find frequent substructures ( subgraphs ) in graph - based mass datasets . graph isomorphism is the problem of deciding whether two graphs have identical structures ( e.g. , finding common fragments from two molecules ) . the second problem , subgraph isomorphism , is the problem of deciding whether one graph is a subgraph of another graph . graph - based frequent substructure extraction methods avoid this high complexity using some limitations . in recent years they rely their approaches on candidate subgraph extraction processes and pruning some of them using some methods or representations . subdue is one of the well - known graph - based frequent substructure mining approaches . simplifying the data by compressing repetitive substructures , subdue method finds frequent substructures within the data . it also enables abstraction of detailed and complex structural data by iteratively constructing a hierarchical description of it . the algorithm first searches a single vertex that matches with the substructure . at each iteration , algorithm expands matching substructure using a greedy selection of best suitable neighbor edges until all possible substructures are found . subdue finds an incomplete set of frequent substructures , because of the greedy idea behind it . some other approaches depend on the principle of a priori algorithm in basket analysis . a priori - based frequent substructure mining methods extract association rules which have higher support and confidence than those of a threshold value . for example , to minimize computation and storage , frequent subgraph discovery ( fsg ) algorithm also uses canonical representations . a graph can be represented with many different canonical labels depending on the orders of vertices and edges . therefore , fsg searches all permutations of vertices to find a unique canonical label . to narrow down the search space , vertex invariants technique that partitions the graph into subgraphs ( not mentioning candidate frequent subgraphs ) fsg generates candidate subgraphs and increases the size of the candidate subgraphs by adding one edge to each candidate at each time . using a breadth - first approach frequencies of achieved candidate subgraphs are calculated to prune the subgraphs that do not satisfy the support constraint . for each candidate , a mapping with the other graph is searched to solve the graph isomorphism problem using canonical representations . graph - based substructure pattern mining ( gspan ) transforms the problem into finding common related parts in depth - first search ( dfs ) codes to find frequent subgraphs . it uses depth - first search tree structure and dfs lexicographic order to find frequent subgraphs . in this approach , each graph has a dfs code , and minimum dfs codes are used for comparison of two graphs . there is a counter in each node that shows the number of graphs including the subgraph . to prune the search space in the tree structure , , it removes nodes which have a counter value smaller than support threshold value . in the second one , it removes nodes which have a number of nodes more than the desired number of nodes . it also uses a tree structure where each node represents a subgraph and uses depth - first search in the tree structure . inductive logic programming ( ilp ) is one of the well - known techniques in graph mining . although many graph - based searching approaches focus only on instances from only one class , ilp uses observations from every class . however , it generally does not support numerical calculations and can not model quantitative classification problems . in this section , we propose a novel approach to discover frequent fragments of active and inactive molecules . previous researches mostly use graph - based frequent substructure mining methods to discover frequent active fragments that account for the activity of the molecules for a specific disease [ 3 , 11 ] . however , these methods work like black boxes where the data with some parameters are fed in the beginning and the solution is outputted at the end . hence , domain experts can not easily interrupt the process to incorporate their knowledge into the solution . this leads to two main problems : ( 1 ) the found solutions should be extensively examined by the domain experts at the end and ( 2 ) any failure in the early stages of the process can not be corrected by the domain experts until the overall process is completed . in this paper , we advocate directly incorporating domain knowledge into the solution process at different levels . for this purpose , we propose to use a data pipelining approach , where domain experts or users can intervene the overall process to enhance the solution with their knowledge and expertise . figure 1 shows the data flow diagram of the proposed data pipelining structure , where raw molecular data from a dataset are filtered iteratively . the dataset contains 3d graph representations of molecules that are either active or inactive with respect to a specific disease , for which a new drug is to be designed . with the proposed data pipelining , a domain expert or a user is able to provide critical information to the system at the intermediate steps . at the beginning using these parameters and the dataset , the system computes two histogram - based visual representations as described in section 3.2 : activity map and inactivity map . before the fragment mining begins , activity and inactivity maps are displayed to the user to show information about candidate frequent fragments for the active and inactive molecules , respectively . if the candidate frequent fragments are not clearly seen from the visual representations , the user can change the parameters . once the resulting maps are satisfactory for the user , infrequent and insignificant substructures in the data are filtered automatically using the proposed approach in section 3.2 . after filtering process then , the proposed method transforms filtered data to atom - bond - atom space , where each point represents features of a bond . these clusters can be determined by a suitable clustering algorithm easily as described in section 3.4 , because the filtering step eliminates insignificant and infrequent fragments that constitute the noise between the clusters in atom - bond - atom space . at the initial step of clustering , the found clusters are converted to the corresponding 3d fragments and visualized to the user . using the advantage of data pipelining structure , the user can change the automatically calculated parameters of the clustering algorithm depending on her / his expertise after analyzing the visualized 3d fragments . as a result , she / he either confirms these clusters or tunes the clustering parameters to force the system to recalculate clusters depending on the new parameter values . once the user confirms the computed clusters , unfiltered data and cluster information are used for fragment discovery as described in section 3.4 . because we use the whole dataset ( unfiltered data ) together with the cluster information , fragment discovery is not significantly affected by the data loss caused by the filtering process . using the proposed pipelining structure , we enable users to enhance the solution with their domain knowledge and expertise . if the users are not confident with their expertise , they can simply confirm the automatically calculated intermediate results ( e.g. , discovered clusters ) . in this case , the overall system works almost like a fully automated process . although edges and nodes are used to represent some properties of molecules , it is inefficient to compare different properties of molecules using 3d graph visualization . in this paper , a transformation from 3d graphs to molecular information visualization is implemented . to make this representation more understandable to the experts , images that display information about molecules are created . to represent molecular graphs , electron - topological matrices of conjugency ( etmc ) are used . in this method , a molecule with n atoms is represented with a number of upper triangular fully weighted etmc matrices , each representing various characteristics of molecules . the main advantages of etmc matrices are that their molecular representation reflect a molecule 's electronic and 3d conformational properties and do not depend on the numbers and types of atoms : ( 1)etmc=[a1,1a1,2a1,n1a1,na2,2a2,n1a2,nan1,n1an1,nan , n ] . in our study , diagonal elements ai , i ( i = j ) of an etmc matrix contain information about electronic properties of atoms , and nondiagonal elements ai , j ( i j ) include information about chemical properties of bonds between the corresponding atoms in etmc matrix representation . if there is no bond , the distance between two atoms is used instead . for various characteristics of molecules ( e.g. , bond properties such as value of wiberg 's index ) , hence , different descriptors of molecules can be examined for their accountability on activity for a specific disease . for all integer values of i and j ( 1 i , j n ) , vectors [ ai , j , min ( ai , i , aj , j ) , max ( ai , i , aj , j ) ] are obtained from etmc matrices . hence , corresponding molecules are split into pieces ( bonds ) , where each bond is represented with a vector including information about the bond ( ai , j ) and two atoms at each end ( ai , i and aj , j ) . these pieces are plotted with point pairs , ( ai , j , min ( ai , i , aj , j ) ) and ( ai , j , max ( ai , i , aj , j ) ) , using a transformation from 3d to 2d coordinate system as shown in figure 2 . thus , points are derived from an etmc matrix and then projected onto 2d cartesian system . in the following section , we propose a visual data mining approach that uses the points derived from etmc matrices . in this section therefore , we enhance the understandability of the molecular information by the experts and enable the experts to compare different molecules easily in terms of their desired descriptors . one of the problems while comparing molecules is that molecules posseses different degrees of flexibility . because of this flexibility , even the topological structure of the same molecule can vary under different conditions . therefore , values from etmc matrices can not be compared in a straightforward manner . to overcome this difficulty first , a 2d image is generated from the etmc matrix of a molecule by dividing x , y coordinate system into regular intervals in order to compose a 2d mesh . for this purpose , two parameters are used : x and y . using the parameter x , x - axis of the coordinate system is divided into the intervals so that the width of each interval is x . the crossing intervals on the coordinate system compose a 2d mesh ( a regular grid ) . in literature , moreover , when finding similar frequent substructures , a general grid structure common for every molecule is needed . however , it is difficult to fix an irregular grid suitable for every molecule , because of the noisy data . hence , not to increase complexity of process , a regular grid structure is used in this work . then , the rectangular regions of the mesh where the points fall are shaded . figure 3 shows an example for the creation of a 2d image from an etmc matrix . in our dataset , we have two different disjoint classes of molecules : active molecules and inactive molecules . using the methods described above , we produce images for each molecule in the dataset . now , using these images , we describe how to compose activity and inactivity maps that show characteristic properties of active and inactive molecules , respectively . that is , each map shows the percentage of bonds that are common for the corresponding class of molecules . hence , the frequent bonds in each class of molecules are seen easily on the related map . for active molecules , this time , shades of the rectangles in the activity map denote percentage of molecules falling into the rectangular cells . for this purpose , we use a 2d histogram - based visualization method that shows frequent active fragments in the molecular dataset as follows . a rectangle ri , j can be considered in the 2d mesh , where 0 < i n and 0 < j m. in order to determine the shade of each ri , j in the activity map , we average the shade of the corresponding rectangles ri , j in the images of the active molecules . in order to average shades , hence , an activity map gives information about the parts of molecules that appear frequently in the structure of active molecules in a better perceivable format . the same methodology is used for creation of the inactivity maps , but this time , images from the inactive molecules are used , instead of images from the active molecules . resulting activity and inactivity maps provide important perceivable information to the experts using a histogram - based data visualization . for example , the activity map in figure 4 shows the rectangles with darker and lighter shades . the dark fragments are the structures that are common to the most of the active molecules , whereas the lighter fragments represent the structures that are not repeating in the active molecules , so those fragments may not be significant for the activity . for various characteristics of molecules , we filter the substructures of active molecules if these substructures fall into the lighter rectangles in the activity map . for this purpose if the dataset is noisy ( i.e , it highly contains infrequent fragments ) , a small threshold value will be chosen ; otherwise , the chosen threshold value will be high . in summary , first an activity map is constructed using all of the active molecules in the dataset , then algorithm determines the insignificant parts of each molecule in the dataset using the decisions of expert ( e.g. , provided threshold ) . lastly , the determined parts are filtered to eliminate redundant and noisy information . alternatively , in respect to her / his domain knowledge and expertise , data falling into some regions on the activity map can be removed directly by the expert . selection of x and y parameters does not affect the extracted active fragments significantly , because the filtered data give only preliminary information about activity and inactivity clusters . nevertheless , several values of the x and y parameters should be tried for the best filtering and the best results . after filtering the data using activity and inactivity maps as described in section 3.3 , redundant and noisy bonds are removed from the active molecules in the dataset . the remaining bonds of the active molecules may contain the ones that compose active fragments , so we mine these bonds to discover these fragments . first , the remaining bonds of each active molecule are transformed into 3d atom - bond - atom space , where features of each bond ( and the atoms at its each end ) are represented as a single point . hence , an active molecule with m remaining bonds after filtering is represented with m points . each point is in the form of ai , j , min ( ai , i , aj , j ) , max ( ai , i , aj , j ) , where ai , j , ai , i , and aj , j are values from the etmc matrix of the molecule and correspond to feature values of a bond and feature values of the atoms connected by this bond , respectively . in this way , bonds of the active molecules are transformed into the 3d atom - bond - atom space . the result is a set of points that are distributed over the space , where the points representing bonds with similar features are in proximity . that is , the points are not distributed randomly in the space , but in a way that groups of points representing bonds with similar properties appear . we name these groups of points as candidate activity clusters , which are used to derive candidates of active fragments . in order to find candidates of activity clusters , we use average - link clustering method . in this clustering method , initially each point in the space is regarded as an individual cluster . that is , two clusters are merged if their distance is smaller than that in a predefined threshold . each of found clusters is composed of points that represent bond patterns of active molecules . however , all of these substructures may not be considered as active fragments , because some of them may also be repeated in the inactive molecules . active fragments should be the substructures that exist frequently in active molecules , but rarely in inactive molecules . this means that we are looking for clusters that are composed of bond patterns that are not repeated in the inactive molecules . therefore , after determining clusters , for each cluster , we compute the percentage of active and inactive molecules that contain the molecular pieces in the cluster . the candidate clusters including higher percentages of active molecule bonds and small percentage of inactive molecule bonds are regarded as activity clusters . bonds falling into active clusters are expected to compose active fragments . in order to show advantage of the proposed filtering method , we show the points extracted from raw molecular data in figure 5 and the filtered molecular data in figure 6 in the 3d atom - bond - atom space . although , figure 5 gives a messy view of the molecular data , figure 6 gives a cleaner view of clusters on the filtered data , because noise and uninformative points that show infrequent substructures ( bonds ) are removed from the data after filtering . in figure 9 , we show the resulting clusters on the filtered data with an example of molecule 's two pieces falling into two activity clusters . as we mentioned before , time complexity of subgraph isomorphism hence , heuristics are used instead of exact algorithms to solve frequent substructure discovery problem in graphs . in this paper this approach is composed of four sequential steps : activity map creation , filtering , clustering , and fragment extraction . the highest complexity belongs to clustering step , where average - link clustering is used . complexity of the used clustering algorithm is o(n m ) , where n is the number of molecules in the dataset and m is the maximum number of atoms in a molecule . therefore , the overall computational complexity of the proposed approach is only o(n m ) . in order to demonstrate our approach better , we design realistic experiments with real - life data and simulations on the synthesized graphs . in our experiments , we have used antituberculosis dataset [ 7 , 8 ] . this dataset is composed of 33 molecules ( 13 active and 20 inactive molecules ) . in order to examine our approach extensively first , in section 4.1 , we show a case study to demonstrate how an expert can use our approach for creating activity and inactivity maps and deriving active fragments interactively . second , using antituberculosis dataset and synthetic datasets , we empirically compare our approach with the well - known approaches from literature : subdue , fsg , gspan , gaston , mofa , and ffsm . we have repeated each experiment 10 times and our results are significant according to 2-tailed t - test with 95% confidence level . third , in section 4.4 , we derive active and inactive fragments from the antituberculosis dataset . then , we use the derived fragments as features and evaluate the performance of well - known classification methods in classifying molecules as active or inactive . intuitively , if our approach is successful in determining active and inactive fragments that account for the activity and inactivity of the molecules , then the classification methods using those fragments as features are expected to demonstrate a good performance . all the experiments of the proposed method are tested on 1.6 ghz intel pentium core ii duo , 1 gb ram running windows vista operating system , and matlab 7.1 . to make the approach easily repeatable , we used prtools toolbox for classification methods . those parameters are selected as x = 0.12 and y = 0.07 by the expert . using those parameters , activity and inactivity maps are created as in figures 7 and 8 , respectively . using those activity and inactivity maps this threshold value is decided by the expert in order to keep more information unfiltered . although it may seem that using a 2d activity maps rather than a 3d representation may cause loss of data . it is important to note that these maps are only used in finding approximate locations of clusters where unfiltered data and the preliminary information about clusters are fully preserved . lastly , using unfiltered data and preliminary information about the clusters , the system finds final active fragments . to visualize 3d topology of active fragments , an active template molecule that is selected by the expert is used . graph - based representation of extracted active fragments on the template molecule is shown at figure 9 . we measure the processing time of each step of our approach during the case study . we tabulate these values in table 2 , where the time consumed during human - computer interactions is neglected . our approach consumes around 20 seconds for clustering , while it consumes around only 2 , 0.5 , and 0.7 seconds for activity map creation , filtering , and fragment extraction , respectively . the overall time from beginning to finding active fragments on the case study ( including the time consumed during human - computer interactions ) is around 11 minutes . in this section , using the antituberculosis dataset , we compare our approach with two well - known approaches from literature : subdue and fsg . these approaches are chosen , because they are often used in literature to find frequent substructures of graphs and molecules . therefore , we have used only the active molecules to determine active fragments by fsg and neglected inactive molecules which cause a great decrease in the success of fsg . in our experiments , we use publicly available original implementations of subdue ( http://ailab.wsu.edu/subdue ) and fsg ( http://glaros.dtc.umn.edu/gkhome/pafi/overview ) in order to increase reliability and repeatability . for the basis of comparisons , we use a set of active fragments ( fa * ) that are determined for antituberculosis using extensive analysis in . in order to compare subdue , fsg , and the proposed approach let fa , fa , and fa represent the sets of active fragments computed by subdue , fsg , and the proposed approach , respectively . then , we compare the most significant fragments in fa , fa , and fa with the ones in fa*. by the most significant fragment , we mean the fragment that has the largest support value , which represents the percentage of active molecules including this active fragment at the dataset . let sa , sa , sa , and sa * denote the most significant fragments in fa , fa , fa , and fa*. for comparison of the methods , we used two measures : recall and precision . recall is defined in ( 2 ) as the ratio of correctly discovered bonds of sa * by an approach . in the equation , f(b , s ) is a function that returns 1 if the bond b is contained by the fragment s ( b s ) ; otherwise , it returns 0 . high recall value of an approach implies that it can correctly find most of the bonds in sa * , which is the most significant active fragment ( 2)recall(x)=bsa*f(b , sax)|sa*| . using only the recall metric , we can not measure success of an approach in determining active fragments , because this measure does not use the excess bonds found by the approach . that is , recall of an approach x is 1 if sa sa * or sa * sa , where x finds also the bonds that are not a part of an active fragment ( sa * ) . therefore , we introduce precision metric in ( 3 ) . the precision value of an approach x is high if all of the bonds in sa are included in sa*. if both of the recall and precision values are close to 1.0 for an approach , this means that this approach can correctly and precisely find most significant active fragments ( 3)precision(x)=bsaxf(b , sa*)|sax| . the table implies that our approach can correctly determine the most significant active fragments , because its recall and precision values are both close to 1.0 . although the performance of subdue is also high , our approach significantly outperforms it . unlike subdue and the proposed approach , the performance of fsg is low ; its recall and precision values are 0.40 and 0.67 , respectively . this performance difference is intuitive , because fsg can not use the class information ( e.g. , active molecule and inactive molecule ) while determining fragments . although fsg can find fragments belonging to active molecules , these fragments may not be active fragments , because they may also repeat in the structure of inactive molecules . however , unlike fsg but similar to the proposed approach , subdue uses class information while determining frequent fragments . hence , it can determine frequent fragments that exist in active molecules but not in inactive molecules . therefore , in our experiments , performance of subdue is higher than the performance of fsg . these findings imply that inactive molecules may have a significant importance on determining active fragments . hence an approach that uses both active and inactive molecules should be used to solve active fragment discovery for drug design . one of the main deficiencies in the methods proposed in graph - based data mining is their narrow view on the problem . however , molecules may have fragments that give them activity with respect to a specific disease , but these fragments may not repeat exactly in each active molecule . instead , these fragments may repeat with some deviations , which implies the necessity of subgraph mining methods that discover not only exactly repeating substructures but also the ones that repeat with some deviations . in this paper , we propose a clustering - based molecular graph mining method for frequent substructure extraction , where pieces ( atom - bond - atom triples ) with similar features fall into the same clusters . this enables frequent substructures to be discovered even though these substructures repeat in molecules with some variations . in this part of our experiments , using synthetic datasets , we show how successful our approach is with respect to others when substructures giving activity to a molecule do not exactly repeat but repeat with some small variations . we compare the proposed approach with parmol ( http://www2.cs.fau.de/forschung/projekte/parmol/ ) package for frequent molecular subgraph mining , which includes publicly available implementations of gspan , gaston , mofa , and ffsm . we compare our approach with these well - known methods using the synthetic datasets . like the proposed approach , a synthetic graph dataset is composed of two sets of graphs : the graphs representing active molecules ( sa ) and the graphs representing inactive molecules ( si ) . in these graphs , labels are real numbers , where the nodes and edges take their labels in the ranges of [ 04 ] and [ 06 ] , respectively . before creating the graphs in sa or si , we first create three different sets of patterns : the subgraphs repeating only in active molecules ( pa ) , the subgraphs repeating only in inactive molecules ( pi ) , and lastly the subgraphs repeating both in active and inactive molecules ( pai ) . each graph g sa is created so that it includes subgraphs from both pa and pai . however , before adding these subgraphs to g , we modify them according to a parameter pn , called probability of noise . this is done because of the fact that a pattern may not exactly repeat in real - life molecular datasets ; instead the same pattern may repeat in different molecules with slight variations . if pn = 0 , then we directly add subgraphs from sa and sa i to g. however , if pn > 0 , then we slightly modify the labels of the nodes and edges in these subgraphs with probability pn by adding some noise , before adding them to g. the amount of noise to be added is determined randomly in the range of [ 00.25 ] . we also add m other nodes to g , where the number m and the labels of these nodes are chosen randomly . in order to ensure connectivity of the graph , we add edges randomly between these nodes and the others . lastly , we randomly give a unique i d to each node in the graph . similarly , we create graphs for inactive molecules , but this time these graphs are produced using the patterns from pi and pai . using this procedure , we compute a number of graphs representing active and inactive molecules . in the resulting graph dataset , some similar patterns repeat in almost every graph , while some others repeat only in the graphs representing either the active molecules or the inactive molecules . there is a similar case in the real - life datasets , where only the substructures repeating in active molecules are responsible for the activity of the molecule , while the substructures repeating almost in every molecule or only in the inactive molecules do not have any significant effect on the activity . we created eight different datasets using different numbers of active and inactive molecules as well as different values for the probability of noise . for each dataset , we exactly know which subgraphs are repeating only in the graphs representing active molecules . note that these subgraphs are not repeating exactly the same but with some small differences ( called noise ) . hence , we can quantitatively measure how successful our approach is , compared to the other approaches ( gspan , gaston , mofa , and ffsm ) , while finding these subgraphs . our experiments show that the proposed approach and the graph mining methods gspan , gaston , mofa , and ffsm can find all of the active substructures correctly when there is no noise ( pn = 0 ) . however , an increase in the probability of noise results in a dramatic performance decrease in the graph mining methods gspan , gaston , mofa , and ffsm . for example , when pn is increased to 0.25 , although their precisions remain 1.0 , the recall values of gspan , gaston , mofa , and ffsm decrease to 0.33 , 0.28 , 0.21 , and 0.19 , respectively . this means that they do not find fragments that are not frequent ( precision is 1.0 ) , but they can only find 33% , 28% , 21% , and 19% of the frequent fragments , respectively . on the other hand , precision and recall of the proposed approach are both 1.0 , which means that the proposed approach can correctly determine frequent fragments even though these fragments do not eactly repeat in molecules , but repeat with some variations ( pn = 0.25 ) . the situation becomes more dramatic when pn becomes 1.0 ; both precision and recall become zero for gspan , gaston , mofa , and ffsm . this means that they can not find any part of the frequent fragments , because nodes and edges in these fragments do not exactly repeat in molecules . in all these cases , our approach can find almost all of the active substructures correctly ; it has precision and recall values almost equal to 1.0 when pn > 0 . for varying size of the datasets , performance of our approach does not change . these experiments show that our approach can successfully find frequent substructures even though these substructures do not repeat exactly in the molecules ; however , the other methods can find these substructures correctly only if they repeat exactly the same in the molecules ( pn = 0 ) . searching a molecular substructure rapidly in a molecular database is an important research problem in drug design . in literature , graph - mining techniques are mostly used to search molecular databases for the new molecules that are likely to be active for a specific disease [ 1 , 21 ] . using classical graph - searching methods , it is difficult to find molecules with specific frequent substructures , because of the time complexity of subgraph isomorphism . instead of using graph - searching methods , classification methods from machine learning literature are also used for the estimation of active and inactive molecules in a molecular database . therefore , the molecular data should be preprocessed before using these methods . in this paper , we propose an approach for deriving active fragments that account for the activity of the active molecules . we can also derive inactive fragments from inactive molecules using the same method . in this case , the derived inactive fragments account for the inactivity of the inactive molecules . then , we can use information about activity and inactivity clusters ( representing active and inactive fragments ) as features to represent each molecule using the same dimensions . let us have n1 activity clusters and n2 inactivity clusters that we derive using the proposed approach . for each molecule , we prepare an array of n1 + n2 dimensions , where each dimension represents one cluster . if the molecule has an active or inactive fragment , the corresponding dimension of the vector is set to the minimum distance from the points of molecules to the corresponding cluster 's center ; otherwise , it is set to 0 . this way , then , we input this training set to different classifiers : lda , k - nn , svm , and decision trees ( dt ) . lastly , using one - leave - out cross validation method , we measure the classification performance of the classifiers . then , for each molecule in the dataset , they compose a feature array of a + b dimensions , where each dimension takes binary values ( 1 or 0 ) to show whether a specific fragment exists in the molecule or not . using the derived arrays as training set , they train a classifier to measure how well the derived fragments can be used to classify molecules as active or inactive with respect to antituberculosis . their activity / inactivity prediction using leave - one - out cross validation is 80% . in order to measure the performance of our approach better , we also train the classifiers with the original unfiltered data that contain not only active / inactive fragments but also other fragments that are filtered out while determining these active / inactive fragments . unfiltered data contain more information about the molecules , so classifiers using the unfiltered data may have a better performance with respect to their performance using only a subset of these data ( i.e. , only active and inactive fragments ) . our main aim in this paper is to correctly determine active / inactive fragments that are responsible for the activity / inactivity of the molecules . if we determine these fragments correctly , the classifiers using only those fragments as features should also demonstrate a good performance in classifying molecules . in table 5 , we tabulate our results for x = 0.12 and y = 0.07 using the original ( unfiltered ) data and the filtered data , where only active and inactive fragments are used as features . our results show that most of the classifiers ( except lda ) achieve the same performance when they use only active / inactive fragments as features ( filtered data ) and when they use the whole molecular data ( unfiltered data ) . in our experiments , the best performance belongs to svm and dt classifiers , which always correctly classify active and inactive molecules ( success is 100% ) . performance of other classifiers is also very good ; almost all of the classifiers can correctly classify molecules in more than 95% of the cases . we immediately recognize that lda can not find active molecules and labels all of the molecules as inactive when unfiltered data are used . the reason behind this is the fact that lda maps all data into a ( c 1)-dimensional feature space , where c is the number of classes . in our case , there are only two classes , which means that lda tries to discriminate these two classes in a 1-dimensional space . since the unfiltered data contain highly overlapping structures and features between active and inactive molecules , classification in a 1-dimensional space is clearly not enough when unfiltered data are used . however , when we filter the data using the proposed method , we remove the common and noisy parts of the molecules that are not significant for being active or inactive . hence , lda can successfully learn active and inactive molecules in a 1-dimensional space using the filtered data . those results imply that our approach can correctly determine the active and inactive fragments , and those fragments can successfully be used as features in classification . moreover , when the classifiers use only the active / inactive fragments extracted from filtered data rather than fragments extracted from original ( unfiltered ) data , it is observed that overall classification process is 2.7 times faster . infectious diseases like tuberculosis are the leading causes of death and suffering worldwide . the world organization of health reports a significant rise in drug resistance of diseases like tuberculosis . this increasing drug resistance highlights the need for new , safer , and more effective drugs . however , designing new drugs is an exhaustive process , where discovery of fragments that account for biological activity and prediction of biological activity in relation to the chemical structures of molecules are crucial . in this paper , we extend our previous studies in . unlike the previous work , this paper discusses the role of human experts in the process , presents computational complexity of the overall approach , analyzes the robustness of the approach to the noise , and lastly compares the proposed approach with current approaches ffsm , mofa , gspan , and gaston in detail using synthetic datasets . first , previous graph - based approaches for frequent pattern mining methods are fully automated and do not allow experts to interact with the system to incorporate their expertise to the process . however , the proposed approach enables experts to interact with the system and improve the solution with their expertise . second , current methods can determine frequent patterns only if these patterns exactly repeat in molecules . however , in many settings , patterns may not repeat exactly , but with some variations . for example , in different conditions ( e.g. , temperatures ) , some features of the bonds and atoms in a molecule may slightly change ( e.g. , orientation of atoms ) ; this means that the same pattern may appear slightly different even in the same molecule under different conditions . while current methods may not determine these patters that appear with slight variation in molecules , our approach successfully determines them using clustering . we have evaluated the performance of our approach using experiments on antituberculosis dataset and various synthetic datasets . our experiments show that our approach can correctly determine active fragments of molecules that account for the activity of those molecules . we also show using our approach that classification methods can achieve good performances while determining biological activity or inactivity in relation to the chemical structures of molecules . in this work , we use antituberculosis dataset in order to demonstrate and evaluate our approach . as a future work ,

designing new medical drugs for a specific disease requires extensive analysis of many molecules that have an activity for the disease . the main goal of these extensive analyses is to discover substructures ( fragments ) that account for the activity of these molecules . once they are discovered , these fragments are used to understand the structure of new drugs and design new medicines for the disease . in this paper , we propose an interactive approach for visual molecule mining to discover fragments of molecules that are responsible for the desired activity with respect to a specific disease . our approach visualizes molecular data in a form that can be interpreted by a human expert . using a pipelining structure , it enables experts to contribute to the solution with their expertise at different levels . in order to derive desired fragments , it combines histogram - based filtering and clustering methods in a novel way . this combination enables a flexible determination of frequent fragments that repeat in molecules exactly or with some variations .

1. Introduction 2. Related Work 3. Interactive Mining of Molecules 4. Evaluation 5. Conclusions

PMC3670211

a single molecule that functions as a molecular conductance switch under external stimuli can be useful for molecular memory and logic devices . a broad range of molecular switches have been reported in the literature , activated with different kinds of external stimuli . light is a very attractive stimulus due to its fast response time and compatibility with already existing experimental setups . the existing switches can roughly be divided into two categories : redox / polarization switches , where the molecule ( or part of it ) takes up or loses an electron , or isomerization switches , where the 3d - structure ( geometry and/or connectivity ) of the molecule is changed . the basic requirements for a molecular reaction to be suitable for switching applications are : ( i ) it must have high difference in the magnitude of the conductance for the on and off states , i.e. , high switching ratio ( sr ) , ( ii ) it must be stable , and ( iii ) it needs a sufficiently high barrier between the on and the off states to hinder accidental ( de)activation . isomerization switches should ideally have the on state represented by a planar compound with a linearly conjugated path with maximal p-orbital overlap . the compound representing the off state , on the other hand , should have a disruption in its p-conjugation , and such a disruption could be accomplished by a saturated molecular segment ( structural isomerization ) or by a large twist in the conjugated path ( stereoisomerization ) . at the same time , for a possible use in a solid state device the length of the molecule should stay essentially the same which has been a major drawback for some of the previously studied molecular photoswitches , i.e. , azobenzenes . further , it should be possible to switch the molecule between the two states with an external stimulus , and most interesting is the possibility to switch in one direction with one stimulus and in the other direction with another stimulus . the -silyl shift for formation of a si = c double bonded compound , a so - called silene , from an acyloligosilane is a structural isomerization that potentially could be used for a novel type of molecular switch . this reaction was used by brook and co - workers to form the first isolable silene in 1981 ( scheme 1 ) . importantly , the acyloligosilanes such as 1a - off and 1b - off were turned into the brook - silenes photolytically through irradiation by mercury spot lamps . however , it was also observed that the silenes rearranged back to the acyloligosilanes ( e.g. , silene 1b - on has a half - life of 15.8 h at 25 c in ether solution ) . silene 1a - on , on the other hand , could be observed spectroscopically ( ir , uv , nmr ) over a period of two weeks at room temperature , during which it gradually rearranged back to 1a - off . with regard to 1a - off/1a - on , we report herein ( vide infra ) the calculated reaction energy for 1a - on formation to be 51.2 kj / mol , and the activation energy for the thermal back - rearrangement from the silene to silane to be 82.1 kj / mol ( scheme 1 ) . in a recent computational study , eklf , guliashvili , and ottosson found silene 1c - on to be 20.1 kj / mol higher in energy then 1c - off , and an activation barrier of 123.4 kj / mol separated the silene from the acyloligosilane . this property of the acyloligosilane / brook - silene system should be possible to elaborate further and potentially exploit in a molecular conductance switch . the described activation energies for thermal back - rearrangements from the silene to the silane to progress at moderately elevated temperatures should be in the range 90120 kj / mol , and the reaction energies for this process should be moderately exothermic . reaction and activation free energies for the thermal processes of 1a computed at the b3lyp/6 - 31g(d ) level as reported herein ( vide infra ) . transition state abbreviated as ts . our earlier computational study showed a distinct difference in the relative energies between the brook - type 1,1-dimethylsilenes as compared to the analogous brook - type 1,1-bis(trimethylsilyl)silenes where the former have higher relative energies by 4060 kj / mol compared to the latter species . this feature is interesting as an acyloligosilane / brook - silene switch should have the silene ( the on state ) slightly higher in energy than the acyloligosilane ( the off state ) so that a driving force for the thermal back - rearrangement is established . consequently , the relative energy of silene versus acyloligosilane can be tuned by a proper choice of substituents at the central si . moreover , a change from a migrating silyl group to a migrating germyl ( stannyl ) group weakens the o e bond strength ( eo e = si > ge > sn ) , raising the energy of the silene ( on ) side . increased steric bulk of the migrating group should also disfavor the acylsilane ( off ) side as recently found by bravo - zhivotovskii et al . finally , one can expect that increased conjugation , as found in the silenes studied herein , will lower the relative energies of these species . our initial model species for the acylsilane / brook - silene switches , together with their carbon analogues , are displayed in scheme 2 . these model silenes have a linearly conjugated path stretching between the two ends of the molecule , whereas the acyloligosilanes have an interruption . we first examine the acyloligosilane / brook - silenes in their trans - conformers with thiol end groups since those ( still ) are ubiquitous anchors to gold electrodes , even though a range of other groups with improved anchoring properties recently have been identified . thus , we report on a new class of constitutional molecular switches based on the -silyl shift going from a silane to a silene . we start by the linear acyloligosilane / brook - silene molecules where we calculate the reaction ( rxn ) and activation energies as well as the transport properties using density functional theory ( dft ) and nonequilibrium green s functions ( negf ) . however , based on our work on these linear switches , we found that it is important to constrain the motion when the system progresses along the reaction coordinate over the transition state ( ts ) . consequently , these first candidates needed to be further modified to perform optimally as molecular conductance switches in solid state devices . the second half of the study is therefore devoted to computational design of a set of more realistic molecular switch candidates . reaction and activation energies were computed for the new cyclic silane / silene switches , where we also find constrained motions . furthermore , transport investigations reveal high switching ratios . using a single - level transport model , we estimate the location of the most dominating level for electron transport ( e0 ) and the broadening of the level ( ) and their influence on the switching ratio . full geometry optimizations without symmetry constraints were carried out with the gaussian09 program package . all geometry optimizations reported were performed with the b3lyp hybrid density functional theory ( dft ) method in combination with the 6 - 31g(d ) valence double- basis set . frequencies were computed to confirm that the calculated structures correspond to local minima or transition states on the potential energy surface . the b3lyp hybrid dft method was chosen as we earlier have found that the deviations in the geometries of a set of 15 small substituted silenes in a total of 27 different conformers , including silenes with natural ( si = c ) as well as reverse ( si = c ) bond polarization , when compared against ccsd / cc - pvtz geometries , are smallest for hybrid dft methods . neither mp2 nor generalized gradient approximation ( gga ) dft methods , as computationally inexpensive methods , lead to better agreements . the m06 - 2x meta - hybrid gga dft method was found to give a slightly better mean absolute deviation but , on the other hand , gave spurious geometry deviations for a few of the test silenes when compared against the ccsd geometries . the effect of the basis set on the calculated geometries was also examined on the test set of small silenes mentioned above , and it was revealed that the mean absolute deviation , as compared to ccsd / cc - pvtz , was 0.017 and 0.019 with regard to the si = c double bond lengths calculated at b3lyp / cc - pvtz and b3lyp/6 - 31g(d ) , respectively . for the 15-off/15-on and 17-off/17-on silane / silene switches , we also examined the quality of the b3lyp/6 - 31g(d ) calculations with regard to basis set and with regard to treatment of sterically congested situations ( dispersion ) . b3lyp calculations with the 6 - 31g(d ) valence double- basis set were compared against b3lyp calculations with the 6 - 311+g(d , p ) valence triple- basis set , and both reaction and activation energies when calculated at the b3lyp/6 - 311+g(d , p)//b3lyp/6 - 31g(d ) level were found to agree well ( see supporting information ) . when the b3lyp/6 - 31g(d ) energies are compared against calculations with the m06 - 2x hybrid meta gga functional , i.e. , a dispersion corrected functional which is better able to describe sterically congested situations , it was also found that the deviations in energies were modest ( see supporting information ) . time - dependent dft ( td - dft ) calculation were carried out with b3lyp in combination with a 6 - 311+g(2d , p ) basis set , considering ten transitions each ( singlets and triplets ) . transport calculations were carried out from first principles with a method based on nonequilibrium green s functions ( negf ) combined with dft as implemented in the transiesta package . the relaxed molecular structures were inserted as their dithiolates between two au surfaces and relaxed once more to optimize the au the device consists of three parts : left electrode , molecule , and right electrode . the electrodes are modeled by six layers of gold atoms where the three outer layers are relaxed , while the others are kept at the experimental bulk positions . in the lateral dimension a 6 6 supercell ( 17.5 17.5 ) was used , large enough to remove interactions between periodic images . all relaxations are performed at the dft level with the siesta package , and core electrons are modeled using troullier - martins soft norm - conserving pseudopotentials . the valence electrons are expanded in a basis set of local orbitals using a double- plus polarization orbital ( dzp ) set for electrons in the molecule and a single- plus polarization orbital ( szp ) for electrons in the gold electrode . the off states of switches 2a , 2b , 3a , and 3b were optimized in the anti - conformers , and the on states were optimized as e - isomers to match the expected structures of a rigidly confined acyloligosilane / brook - silene switch . for these species , it becomes clear that for the two acyloligosilane / brook - silene pairs the acyloligosilane and the silene side are nearly isoenergetic ( see table 1 ) , while for the all - carbon -silyl ketone / siloxyalkene pair the alkene is much lower in energy . the very large difference in relative energy for the two sides of the all - carbon pairs can not be changed significantly through substitution , and our calculations thereby reveal why the -silyl shift to form an alkene , and the reverse reaction , are not suitable to function as a reversible molecular switch . the -shift of a silyl group from the -carbon of a ketone to the carbonyl oxygen is thus a purely hypothetical molecular switching reaction . however , with a suitable tailoring of the acyloligosilane / brook - silene switch pair , it should be possible to design a system with the silene slightly above the acyloligosilane in energy and which has an activation energy for the thermal back - reaction which is reasonably high ( 90120 kj / mol ) so that moderately elevated temperatures can be used to turn off the switch . the activation energy for the -silyl shift from 1a - off to 1a - on is calculated to be 133.3 kj / mol at the b3lyp/6 - 31g(d ) level , but the activation energy for the back - reaction taken relative to 1a - on is only 82.1 kj / mol . as this silene gradually rearranged back to the silane during a period of two weeks , it is obvious that the barrier for a silane / silene switch needs to be slightly higher . free energy of activation relative to the off - state ( normal print ) [ kj / mol ] . free energy of activation relative to the on - state ( in italics ) [ kj / mol ] . restricted ss distance . importantly , the ss distances within each of the four pairs stay essentially constant when going from the off structure to the on structure ; however , investigations of the transition states revealed that the migration of the silyl group requires a significant rotation around the central si this rotation induces a large motion of the two thiol anchors during the ( thermal ) rearrangement and consequently a significant shortening of the ss distance to 9.57 for 2b - ts , i.e. , a reduction by approximately 4 . attempts to mimic the rather fixed distance situation , as would be seen in a solid - state setup , by virtually fixing the ss distance to 13.54 resulted in a large increase in the activation energy from 115.7 to 190.0 kj / mol of 2b - ts , a prohibitively high activation barrier for thermal back - rearrangement at reasonably elevated temperatures . brook and co - workers used acyloligosilane 1a - off ( scheme 1 ) to form silene 1a - on , which has uv / vis absorption maxima at 348 nm ( = 100 ) and 339 nm ( = 5200 ) , respectively , showing good agreement with td - dft calculated values of 362 nm ( oscillator strength f = 0.0118 ) for 1a - off and 351 nm ( f = 0.2165 ) for 1a - on . the presently investigated acyloligosilanes 2a - off and 2b - off have their computed lowest uv / vis absorptions with significant calculated oscillator strengths at 395.7 nm ( f = 0.1194 ) and 381.3 nm ( f = 0.0352 ) , respectively ( see supporting information ) . from this we can conclude that the present silane / silene pairs could form the basis for a light - driven molecular switch under the condition that the motion along the reaction path is constrained . to estimate the switching properties of the acylsilane to silene transition , we performed first - principles dft calculations for the acetylenic compounds ( 2a/3a ) shown in scheme 2 . compounds ( 2b/3b ) showed a different behavior in the electrode setup which is briefly discussed in the supporting information . the current response for bias voltages of 0.1 , 0.2 , 0.5 , and 1.0 v are shown in figure 1 . looking at trends in the current response , both compounds 2/3a - off and 2/3a - on show a similar behavior with a linear increase of the current for low voltages and more rapidly increasing current at higher voltages . to investigate their performance as switches , we calculate the on / off switching ratio of the current for both the zero - bias limit as well as for a bias of + 1 v. due to the unsymmetric nature of these molecules , we also investigate if they have a rectifying property on the current , hence if they could function as diodes . the on / off ratio is higher for compound 2a compared to the all - carbon switch 3a . from figure 1 it can be seen that this is due to a more conducting off state for 3a , while the current response is very similar for the two on states . when going to higher biases , the switching ratio decreases due to the stronger current response from the low - conducting acyloligosilanes and ketones when the bias is increased . calculated current voltage ( i v ) characteristics for off and on state structures of compounds 2a and 3a , respectively . note the different scales for the calculated currents in the on and off states . rectifying ratio , calculated as the ratio between the currents at bias voltages of + 1 and 1 v. to explore the physical mechanisms behind the switching , we start by calculating the partial density of states ( pdos ) for the junctions with 2a under equilibrium conditions . the density of states spatial distribution can be calculated by summing up the states in the transverse direction and normalizing to the atomic density . this gives the number of states/(ev atom ) along the transport direction which is shown in figure 2 , where the x - axis is the coordinate along the transport direction and the y - axis shows the energy ( e ef ) for the state , where the magnitude of the dos is indicated by the color , from blue ( low ) to red ( high ) . space resolved normalized density of states at zero bias for the junctions with compound 2a . the off state is shown in the top panel and the on state in the bottom panel . the space resolved density of states have the transport coordinate on the x - axis and the energy of the state on the y - axis . the color scale goes from blue ( low density of states ) to red ( high density of states ) . for 2a - off the homo of the molecule is located about 0.5 ev below the fermi level ( ef ) of the electrodes and is not so well coupled to the electrodes . the electronic states in the molecule that are induced by the interaction with the metallic electrodes are often referred to as metal - induced gap states ( migs ) . migs can be seen in the spatial dos plot as light - blue protrusions penetrating into the molecule from both electrodes but rapidly decaying toward the center of the switch . the -silyl shift leads to the formation of 2a - on ( 3a - on ) having a si = c ( c = c ) double bond . the dos spatial distribution for 2a - on ( bottom panel ) shows that the homo of the molecule is shifted closer to ef , and at the same time it is delocalized over the molecule and coupled to both electrodes . coupling of the homo to the electron states in the electrodes causes broadening of the dos peak . we see two light blue satellites of the homo slightly above and below in energy in the dos spatial distribution plot created by the extended migs overlapping with the broad homo peak . the broadening and delocalization of the homo and surrounding migs create a relatively smooth dos close to the fermi level . this density is responsible for the absence of current onset around the position of the homo ( 0.2 or 0.4 v bias ) , when increasing the bias voltage for 2a - on ( 3a - on ) . furthermore , we show the wave function of the most conducting scattering state at zero bias in figure 3 . the isosurface of the scattering state has different colors depending on the phase ( + 1 , 1 , + i , i ) as explained in the figure caption . the magnitudes of the isosurface lobes indicate where the electrons incoming from the left electrode are traveling since the absolute square of the wave function corresponds to the density of traversing electrons . for a wave , incoming from the left and which is mostly reflected , the real part will show an exponential decay into the junction from the left , while the imaginary part will be very small and in most cases not visible ( if the same isovalue is used when plotting ) . as in the case of dos spatial distribution for 2a - off , the scattering state ( top panel ) shows a very weak coupling through the central si c bond , where the real part of the state decays rapidly from the left into the junction indicating that in this case we have a poor conductor acting as a tunneling barrier . the imaginary part of the wave function is not visible due to the low transmission of the channel and that the wave function is chosen to be real at the incoming side . visualization of the most transmitting eigenchannel wave function ( incoming from the left electrode ) at the fermi energy for the junctions with compound 2a . off state shown in the top panel and on state in the bottom panel . the isosurfaces of the eigenchannel wave function are colored according to phase and sign , the positive / negative real part being colored in deep - purple / light - blue ( the imaginary part is below the cutoff value and not visible ) , and both junctions plotted with the same isovalue to be comparable . from the scattering state of 2a - on ( bottom panel ) the si = c double bond formed from the -silyl shift shows a good connection through the central bridge providing a higher conductance through the migs at the fermi level . it is still almost only the real part of the wave function that is visible , but it does not decay as fast into the junction . from the shape of the lobes we can see that the main transport goes through the p-orbital system of the molecule . similar conclusions can be drawn for 3a ; i.e. , for the on state we have a very good linear conjugation through the complete molecule , while for the off state the conjugation is broken in the central unit due to the si c ( c c ) single bond ; hence , the transmission drops significantly . even though the studies of reaction thermodynamics as well as electron transport of 2a , b reveal that these compounds exhibit the desired properties , the large structural change along the reaction pathway led us to a redesign . in particular , the twisting about the central sic bond that takes place during the -silyl shift , leading to drastic variations in the ss distance during the reaction , needs to be reduced , and a silane / silene unit with less conformational flexibility is required . this can be accomplished by introduction of a fused cyclic system that would prevent large motions leading to / from the ts . however , this restricted system must not have a raised energy of the ts state as this would hamper a thermal back - reaction . to provide a cyclic system with an overall p-conjugated pathway in the on state , we designed a five - membered ring with a central sic unit having a si = c double bond and two neighboring exocyclic c = c double bonds . to accommodate for steric congestion , either a benzene or a phenylacetylene group is attached to the double bonds in an all - trans arrangement . to provide suitable anchors , we kept the thiol groups in para - positions of the phenyl moieties . our initial studies on the linear silane / silene systems indicate approximately the desired thermodynamic and kinetic properties for a light - triggered on switch with a back - reaction which is thermally feasible . one advantage of this rearrangement is the possibility of tuning both thermodynamics and kinetics by slight variations . for our further investigations on the cyclic systems , we focused on three parameters : the nature of the migrating group ( silyl or germyl ) , the steric bulk of the migrating group ( eme3 or ebu2me , where e = si or ge ) , and the acceptor group ( a = o or = nme ) ( figure 4 ) . suggested cyclic molecular conductance switches with different acceptor moieties and migrating groups . a = nme or o , and r = sime3 , sibu2me , geme3 , or gebu2me . blue bars denote n acceptor groups ( a = nme ) , while red bars denote the oxygen acceptors ( a = o ) . simple variation of these three parameters gives a tremendous impact on the reaction thermodynamics ( table 3 ) . while the silyl migrating groups in combination with an oxygen acceptor favor the on state ( from 1.1 to 52.5 kj / mol ) , migration to the nitrogen - based acceptor ( an imine in the off state ) is strongly dependent on the steric bulk of the silyl group ( figure 5 ) . changing the central atom of the migrating group from silicon to germanium favors the off state by up to 76 kj / mol , depending on the steric demand of the alkyl substituents and the acceptor group . however , a general trend for the two different acceptor groups was not found . we further investigated the transition states for the thermal rearrangement of the cyclic silane / silene combinations with the most promising thermodynamic properties . introduction of the five - membered ring results in a reduced ts motion and , moreover , provides suitable kinetic parameters . the activation energies relative to the off states are 126160 kj / mol . the on states are in most cases higher in relative energy , which will allow for a thermal back - rearrangement at ( slightly ) elevated temperatures as the activation free energies for all except one system are gathered in the range 108144 kj / mol . the drawbacks of the linear systems , i.e. , the large motion leading to / from the ts , were also successfully circumvented by the cyclic silane / silene switches . the largest absolute displacements were calculated to be in the range 0.22.0 , which corresponds to relative motions of less than 10% of the absolute ss distance . we assume that these small changes can easily be balanced by lateral movement on the gold substrates , without significant changes in reaction energies . free energy of activation relative to the off state ( normal print ) [ kj / mol ] . free energy of activation relative to the on state ( in italics ) [ kj / mol ] . to prove the viability of photochemically induced silene formation we also performed td - dft calculations for the extended cyclic systems 16 and 17 in their on and off configurations . for compound 16-off , the first allowed uv / vis transition should occur at around 416 nm ( f = 0.0424 ) with a stronger absorption at 404 nm ( f = 0.2193 ) . similarly , but slightly red - shifted , we found for 17-off a weaker ( 417 nm , f = 0.0342 ) and a stronger ( 407 nm , f = 0.1301 ) transition . the extension of the p-system in the corresponding silenes , 16-on and 17-on , is also clearly reflected in the calculated uv / vis spectra of compounds 16-on ( 556 nm , f = 1.1302 ) and 17-on ( 544 nm , f = 0.9959 ) as shown by their longest wavelength absorptions . these long - wavelength absorptions for the on states potentially allow the suggested switches to operate in a reversed manner , in which the on states might rearrange to the corresponding silanes by irradiation with visible light . however , 16-on and 17-on also absorb at the shorter wavelengths , and this feature may potentially complicate the formation of the silenes , which when formed can absorb the irradiation used for their generation and photorearrange back to the silanes . yet , detailed computational and experimental studies and optimization of the photochemistry of the cyclic silane / silene switches are outside the scope of the present investigation . for example , for an optimal silane / silene switch the position of the photostationary state needs to be optimized so that predominantly the silene is formed upon irradiation at the chosen wavelength . the current voltage characteristics have been examined by dft - negf calculations , and due to computational cost we have chosen to study the compounds 4 , 12 ( o - acceptor and trimethylsilyl migrating group ) , 8 , 16 ( nme - acceptor and trimethylsilyl migrating group ) , and 17 ( nme - acceptor and simebu2 migrating group ) here . the current voltage results and their switching ratios are shown in figure 6 and table 4 , respectively . compared to the previous linear compound , we find larger switching ratios at zero bias . by introducing the cyclic unit interference effects , similar to those observed by solomon et al . in phenyl rings , the interference effects can clearly be seen in the transmission spectra where there are several pronounced dips for the off compounds with the phenylacetylene group ( cc ph ) shown in the top panel of figure 7 . for the shorter compounds ( 4 , 8) transmission in the -system of the molecules can now start to play an important role , and this can hide interference effects in the p-system . in the top ( bottom ) panels of figure 8 the local currents in compound 16 are shown for the off ( on ) state . for the off state we find that the interference arises due to ring currents in the ring segment with three saturated carbon atoms . on the other hand , for the on state we observe an undisturbed current path which follows the p-conjugated path through the molecule . conductance on / off switching ratio . rectifying ratio , calculated as the ratio between the current at bias voltage + 1 and 1 v. distance between the most conducting level and ef ( v = 0 ) . proportionally factor for movement of the conducting level under bias ( 0 = e0 ( v/ ) for v , + = r/l ) . a measure of the unsymmetry of the effective coupling to the electrodes . markers denote values obtained from the dft - negf calculations , while the full lines are obtained from the model calculations . note the different scales for the calculated currents in the on and off states . transmission spectra , at zero bias ( v = 0 ) , for the off compounds ( top panel ) and the on compounds ( bottom panel ) . local currents in compound 16-off ( top panel ) and 16-on ( bottom panel ) , where the cross - sectional area of the cylinder is proportional to the current density . red currents represent positive transport direction , and blue currents represent negative direction . the currents are calculated at the fermi energy ( ( l + r)/2 ) , but a small positive bias voltage is assumed for calculation purposes . for a full derivation see okabayashi et al . and paulsson and brandbyge . at higher bias , the switching ratio drops to moderate levels for the cyclic compounds similar to the linear analogues . as in the case of the reaction energies we see that the nme acceptor groups ( compounds 8 , 16 , 17 ) are the most promising candidates with switching ratios on par or higher compared to other compounds studied here and in previous investigations . introduction of the bulkier migration group ( simebu2 , compound 17 ) not only will provide higher steric protection and hence higher kinetic stability but also enhances the switching ratio . the on compounds have rectification ratios ( rr ) close to unity , which is beneficial for practical applications since there will be no directional dependence when running a current through the switch . on the other hand , some of the off compounds have large rr ( especially compounds with o - acceptor , 4 and 12 in table 4 ) , opening the possibility to use them as diodes . for switch applications , the rr of the off compound is not important since there should be no ( or only a negligible ) current running through them , but it will give a difference in sr between + 1 v and 1 v ( here , for rr > 1 , the sr at 1 v will be larger than the sr at 1 v ) . we apply a simple single - level transport model to investigate the influence of the effective coupling ( l,r ) to the left and the right electrode , and the distance between the closest molecular orbital ( e0 ) and ef of the electrodes on the current . from the landauer formula the transmission is obtained as1 we started by considering a static level and symmetric coupling to the electrodes as the earlier literature suggested . , we had to expand the model with a level that could move with the potential level in the electrodes under bias ( 0 = e0 ( v/ ) for v , see table 4 for values of ) and nonsymmetric coupling to the electrodes . the parameters obtained from the model are given in table 4 , showing that the unsymmetry of the coupling ( l/r ) we find from our model agree reasonable well with the rr of the molecules . the results from the model are shown in figure 6 ( full lines ) , together with the dft - negf results ( markers ) . as can be seen from the figure , this extended model can explain our results reasonably well . from this model we can locate the most conducting mo at about 0.5 ev below ef for the off states and almost resonant ( 0.050.15 ev below ef ) for the on states . this can be compared with the transmission spectra at zero bias ( shown in figure 7 ) from the dft - negf calculations , and we find that the peaks in the spectra are located at similar values as obtained from the model ( a comparison between transmission spectra at different biases from dft - negf and our model is shown in the supporting information ) . from the model results we are able to deduce the important parameters affecting the sr . in figure 9 the magnitude of the sr ( symbolized by the size of the filled circles ) is shown as a function of the change in coupling to the electrodes ( = ( lr ) ) and the change in distance between the closest mo and ef . the switching occurs due to either a change of the distance between the closest mo and ef , a change in the coupling between the mo and the electrodes , or a combination of both . to have a large sr ideally the molecule should be in the top right corner of figure 9 , hence it is favorable to have both a large change in distance between ef and the closest mo and a large change in the coupling between the electrodes and the mo . magnitudes of the switching ratio ( sr ) as a function of the change of the closest mo ( e0 ) and the coupling between the mo and the electrodes ( ) . the behavior of the compounds studied here is different compared to the photoswitches studied previously by kim et al . for which they obtained switching due to the change either in coupling to the electrodes or in the relative position of the mo . by the possibility to change both the coupling and the relative position of the mo , as in the case for compounds 4 and 17 , we can obtain a higher switching ratio compared to previous studied molecular photoswitches . at the same time thus , they should represent good candidates of a new set of molecular conductance switches . as pointed out by our criteria to a molecular switch , stability plays an important role , which is especially relevant to the suggested silane / silene based systems . it is known that silanes ( off states ) are persistent species at ambient conditions and could easily be inserted between two gold electrodes prior to switching applications . however , the silenes ( on states ) are reactive , moisture sensitive , and have a high dimerization aptitude . the situation can be significantly changed when the silanes are immobilized between the gold electrodes , and provided the concentration of silanes on the surface is low , the resulting spatial separation between the silenes when formed during switching will inhibit dimerization as a possible decomposition pathway . moreover , the environment on the gold surface around the silane / silene switch can be covered by unreactive alkanethiols or alkanedithiols , e.g. , as recently developed by us in a au nanoparticle / nanoelectrode setup . the dimerization aptitude of the silenes may also be reduced through a range of bulky substituents . the moisture sensitivity , on the other hand , is delicate , and the acylsilane / brook - silene must likely operate under moisture - free conditions . however , reverse polarization , i.e. , as observed for oxygen- or amino - substituted systems , reduces moisture sensitivity of these silenes and might significantly increase the stability of the suggested molecular switches . for example , we found recently that 2-amino-2-siloxysilenes , which are transient silenes extensively influenced by reverse si = c bond polarity ( si = c ) , are unreactive to alcohols . it has also been observed that 1-silaallenes , i.e. , si = c double bonded compounds with an inherent si = c polarization influence , become resistant to water when they bear substituents with large steric bulk . in conclusion , we have introduced a new class of synthetically realistic compounds that can perform as molecular conductance switches which could be triggered by light and/or thermal energy . moreover , we were able to confirm that these switches fulfill the criteria for a useful reversible molecular switch , as given in the introduction , i.e. , a high on / off conductance ratio , high enough barrier separating the two states , and a possibility to realize the switch in a solid state device . the reaction investigated is the -silyl ( germyl ) shift from a low - conducting silane to a conducting silene , and the charge transport characteristics are investigated using dft - negf calculations and a simple landauer model . we started with a set of linear molecules and found by dft - calculations that they can be switched photochemically in one way and switched back thermally . the single - molecule conductance showed good switching ratios at low bias ; however , it was also found that large length variations when progressing along the reaction coordinate , especially at the ts , hamper their potential use as a solid state device . to overcome this last issue we propose a cyclic central unit of the molecule reducing the flexibility while at the same time keeping the switching properties of the linear molecules . calculations further show that we can tailor the reaction dynamics of the compounds , in terms of both reaction barrier ( kinetics ) and energy ( thermodynamics ) between the two states by changing the acceptor unit and/or the migration group . we find that the switching ratio is increased going from the linear molecules to the cyclic , and it is 200 or greater for all considered molecules at zero - bias voltage . we have further analyzed the transport mechanism using the most conducting scattering state , the density of states in the junction , and a single - level landauer model . we find that for the off states we have a mo localized in the central part of the molecule about 0.5 ev below ef . we also see interference effects in some of the off states decreasing the transmission further . for the on states , on the other hand , we have a delocalized -electron system spanning the whole molecule and metal - induced gap states at ef increasing the effective coupling to the electrodes . the most conducting mo is now ( almost ) resonant , enhancing the conductance significantly . these results should be of great interest for the development of a solid state switch or memory device with a high on / off conductance ratio and the possibility to be switched by two different stimuli ( photochemically and thermally ) . with this study we propose a rearrangement and a class of compounds which are completely new to the field of molecular electronics , thus expanding the pool of compounds used in this research area .

on the basis of first - principles density functional theory calculations , we propose a new molecular photoswitch which exploits a photochemical [ 1,3]-silyl(germyl ) shift leading from a silane to a silene ( a si = c double bonded compound ) . the silanes investigated herein act as the off state , with tetrahedral saturated silicon atoms disrupting the conjugation through the molecules . the silenes , on the other hand , have conjugated paths spanning over the complete molecules and thus act as the on state . we calculate on / off conductance ratios in the range of 1050 at a voltage of + 1 v. in the low bias regime , the on / off ratio increases to a range of 2001150 . the reverse reaction could be triggered thermally or photolytically , with the silene being slightly higher in relative energy than the silane . the calculated activation barriers for the thermal back - rearrangement of the migrating group can be tuned and are in the range 108171 kj / mol for the switches examined herein . the first - principles calculations together with a simple one - level model show that the high on / off ratio in the molecule assembled in a solid state device is due to changes in the energy position of the frontier molecular orbitals compared to the fermi energy of the electrodes , in combination with an increased effective coupling between the molecule and the electrodes for the on state .

Introduction Computational Methods Results and Discussion Conclusions and Outlook

PMC4015686

because they are sessile , plants have evolved to respond adaptively to their ever - changing light environment by photoreceptor - mediated physiological responses . plants possess several families of photoreceptors that can sense light direction , duration , quality , and quantity , including red and far - red photoreceptors ( phytochromes ) and three classes of uv - a / blue photoreceptors ( cryptochromes , phototropins , and members of the zeitlupe family ) . these photoreceptors use light signals to regulate almost every phase of plant growth and development , from seed germination through flowering and senescence . for example , phototropins mediate a number of blue light responses in arabidopsis thaliana , including phototropism , chloroplast movement , leaf flattening , leaf positioning , stomatal opening , and rapid inhibition of growth of dark - grown seedlings , thus optimizing light capture for plant photosynthesis and growth . fifteen years ago , christie et al . first demonstrated that phototropin 1 ( phot1 ) acts as a blue light / uv - a receptor for phototropism in land plants . this membrane - associated photoreceptor was the first of a family of two ( phot1 and phot2 ) , and a great deal has been learned about its structure and photochemistry . its two flavin - binding domains ( designated lov1 and lov2 ) have been extensively studied by flash photolysis , ftir , resonance raman spectroscopy , and absorption and fluorescence spectroscopy , among other biophysical techniques . with the discovery that lov domain - containing proteins are widespread in all major groups of bacteria ( including archaea ) , all three major groups of fungi , and all orders of green plants , the lov1 and lov2 domains from phot1 of avena sativa ( domestic oats ) have served as prototypes for investigating lov domains from a wide range of organisms . the structures of several lov domains have been obtained by x - ray crystallography , and a solution structure of oat phot1 lov2 has been obtained by nmr . these combined studies indicate that lov domains consist of five antiparallel sheets separated by short helices . downstream of lov2 is an amphipathic helix ( designated the j- helix ) . on photoexcitation of the flavin chromophore , the j- helix is released from the sheets and loses its coiled structure , and the structural change presumably activates the downstream kinase moiety in phototropins . substitution of hydrophilic amino acids on the hydrophobic side of the j- helix leads to constitutive activation of the kinase function . indeed , the single lov domain in aureochrome from the stramenopile algae actually activates a bzip domain that is upstream of the lov domain , not downstream . hence , photoexcitation of lov domain proteins can lead to more than one type of conformational change in the protein . although much is known about the biochemical and photophysiological properties of the phototropins , progress has been considerably slower in elucidating the post - translational modifications of these photoreceptors . it has been known since the earliest studies that light activates the phosphorylation of multiple sites on a protein that was subsequently identified as phot1 . salomon et al . showed that the phosphorylation was in some way hierarchical , with certain sites phosphorylated at low blue light fluences and other sites phosphorylated only at higher blue light fluences . the same study demonstrated the reverse pattern during a period in darkness as the phototropin returned to its dark state after irradiation . more recently , two different studies identified a number of specific sites that become phosphorylated upon light activation . finally , roberts et al . demonstrated that phot1 from arabidopsis is monoubiquitinated in vivo in response to low fluences of blue light and multi- and/or polyubiquitinated in response to high fluences of blue light . any further characterization of the full - length phototropins has been severely hindered by the lack of success in producing sufficient amounts of highly purified photoreceptors for structural , biochemical , or biophysical studies . here , we use two - dimensional difference gel electrophoresis ( 2d dige ) to examine any dynamic changes in mass or charge occurring in full - length arabidopsis phot1 in vivo during photoexcitation and subsequent dark recovery . we characterize phot1 in its stable dark state as well as its state immediately after saturating light treatment and the completion of phosphorylation . in addition , we employed a combination of immunoprecipitation and mass spectrometry analysis to search for any additional post - translational modifications that have not been previously identified . in this study , a. thaliana columbia ( col-0 ) seedlings and transgenic arabidopsis expressing phot1gfp in a phot15 background were used . for etiolated seedlings , seeds were surface - sterilized and sown on ms plates ( half - strength ms medium , 0.8% agar , 43.8 mm sucrose , ph 5.7 ) , cold - treated ( 2 days at 4 c ) in the dark , exposed to white light of medium intensity ( 100 mol photons m s ) for 6 h , and then incubated in the dark growth room for 4 days at 22 c . blue light irradiation was performed in a growth chamber ( e-30 led , percival scientific , perry , ia , usa ) with far - red , red , and blue ( 468 nm ) light - emitting diode sources . the fluence rate was measured using a li-250a light meter with a li-190sa quantum sensor ( li - cor , lincon , ne , usa ) . etiolated seedlings were irradiated for up to 60 min with continuous blue light ( 20 mol m ) or left in darkness as controls . the whole seedlings , including the cotyledons , hypocotyls , and roots , were collected and frozen immediately in liquid nitrogen until protein extraction and proteomic analyses were performed ( figure 1 ) . schematic documentation of the experimental design ( a ) and a photograph of a representative etiolated arabidopsis seedling ( b ) . four - day - old etiolated seedlings of arabidopsis thaliana ( b ) were used and were either left in darkness ( control ) or irradiated with blue light . thereafter , entire seedlings were used for protein extraction and proteomic analyses , which were combined with mass spectrometric and immunoblot analyses . note that blue light treatment was from the top . the white bar is 1 mm in panel b. microsomal proteins were extracted as described in previous studies . briefly , liquid - ground tissue powder was mixed with three volumes of extraction buffer ( 50 mm hepes , ph 7.5 , 0.33 m sucrose , 5% ( v / v ) glycerol , 3 mm edta , and 5 mm dtt ) with phosphatase and protease inhibitors ( 10 mm sodium fluoride , 10 mm sodium molybdate , 10 mm imidazole , 1 mm activated sodium vanadate , 7 m e-64 , 1.5 m bestatin , 2 m pepstatin , 4 m antipain , and 1 mm pmsf ) and then centrifuged at 10 000 g for 20 min to remove cell debris . the resulting supernatant was centrifuged at 200 000 g for 60 min to pellet the microsomal fraction . the microsomal pellet was dissolved in sds extraction buffer ( 100 mm tris - hcl , ph 8.0 , 2% sds , 1% -mercaptoethanol , 5 mm egta , and 10 mm edta ) and further purified with a modified phenol the protein extract was dissolved in 2d dige buffer ( 6 m urea , 2 m thiourea , and 4% chaps ) and quantified using the bio - rad protein assay . twenty micrograms of microsomal proteins from dark - control or blue light - treated plants ( ph 8.5 ) were mixed with 80 pmol of cy3 or cy5 dyes and incubated on ice for at least 2 h in the dark . cy3- and cy5-labeled proteins were then combined and used for isoelectric focusing ( ief ) . ief was performed on 24 cm ipg strips , ph 310 nl ( ge healthcare , piscataway , nj , usa ) . the running conditions were as follows : rehydration for 2 h , 50 v for 10 h , step and hold at 500 v and then 1000 v for 1 h each , gradient to 8000 v over 3 h , and then at 8000 v until reaching a total of 56 000 v h. second - dimension electrophoresis was performed using 10% sds - polyacrylamide gels . the electrophoresis was performed at 40 v for 2 h and then at 120 v until the bromophenol blue front reached the bottom of the gel . images of cy3- and cy5-labeled proteins were acquired using a typhoon trio scanner ( ge healthcare ) . the estimated ph ranges following ief are indicated in figures 24 . a differential in - gel analysis module with an estimated spot number of 5000 was used for spot detection , and a biological variation analysis module was used to identify spots differentially regulated by blue light ( p value < 0.05 ) . four biological replicates were used in each comparison , and spots of interests were manually checked to confirm spot matching between different gels and to remove artifacts . spot picking and reverse - phase liquid chromatography electrospray tandem mass spectrometry ( lc ms / ms ) analyses were performed as follows . approximately 400 g of protein was labeled with 40 pmol of cy5 dye and separated by 2-de . after electrophoresis , 2-de gels were stained with deep purple stain ( ge healthcare ) . upon scanning the gels with typhoon trio , spots of interests were selected with decyder software and picked by an ettan spot picker ( ge healthcare ) . the excised protein spots were washed twice with 50% acetonitrile in 25 mm ammonium bicarbonate ( nh4hco3 ) , vacuum - dried , rehydrated in 10 l of digestion buffer ( 10 ng/l trypsin in 25 mm nh4hco3 ) , and covered with a minimum volume of nh4hco3 . after overnight digestion at 37 c , peptides were extracted twice with a solution containing 50% ( v / v ) acetonitrile and 5% ( v / v ) formic acid . the extracted digests were vacuum - dried and resuspended in 10 l of 0.1% formic acid in water and then analyzed by lc the digests were separated by nanoflow liquid chromatography using a 100 m 150 mm reverse - phase ultra 120 m c18q column ( peeke scientific , redwood city , ca , usa ) at a flow rate of 350 nl / min in an agilent 1100 high - performance liquid chromatography system ( agilent technologies , inc . mobile phase a was 0.1% formic acid in water , and mobile phase b was 0.1% formic acid in acetonitrile . following equilibration of the column in 2% solvent b , one - half of each digest ( 5 l ) was injected , and then the organic content of the mobile phase was increased linearly to 40% over 30 min and then to 50% in 3 min . the liquid chromatography eluate was coupled to a microionspray source attached to the mass spectrometer . the following mass spectrometers were used for the analysis of the samples : qstar pulsar ( applied biosystems / mds sciex , south san francisco , ca , usa ) , qstar elite ( applied biosystems ) , ltq - ft icr ( thermo scientific , san jose , ca , usa ) , and ltq orbitrap xl ( thermo scientific ) . peptides were analyzed in positive ion mode and in information - dependent acquisition mode to switch automatically between ms and ms / ms acquisition . for experiments using the qstar pulsar and qstar elite , ms spectra were acquired for 1 s in the m / z range between 310 and 1400 . ms acquisitions were followed by 3 s collision - induced dissociation ( cid ) experiments in information - dependent acquisition mode . for each ms spectrum , the most intense multiply charged peaks over a threshold of 30 counts were selected for generation of cid mass spectra . the cid collision energy was automatically set according to the mass - to - charge ( m / z ) ratio and charge state of the precursor ion . a dynamic exclusion window was applied that prevented the same m / z from being selected for 60 s after its acquisition . for experiments using the ltq orbitrap xl and ltq ft icr , ms spectra were acquired in profile mode using the orbitrap or icr analyzers in the m / z range between 310 and 1600 . for each ms spectrum , the six most intense multiply charged ions over a threshold of 400 counts were selected to perform cid experiments . cid collision energy was automatically set to 35% . a dynamic exclusion window of 1 da was applied that prevented the same m / z from being selected for 60 s after its acquisition . peak lists from files acquired by the qstar instruments were generated using mascot distiller version 2.1.0.0 ( matrix science , boston , ma , usa ) . parameters for ms processing were set as follows : peak half - width , 0.02 ; data points per dalton , 100 . parameters for ms / ms data were set as follows : peak half - width , 0.02 ; data points per dalton , 100 . in the case of data acquired in the ltq orbitrap xl and ltq ft thermo scientific instruments , peak lists were generated using pava in - house software , which is based on the rawextract script from xcalibur v2.4 ( thermo fisher scientific ) . in all cases , the peak lists were searched in - house using proteinprospector version 5.4.2 ( the public version is available at http://prospector.ucsf.edu ) . the enzyme specificity was set to trypsin , and the maximum number of missed enzyme cleavages per peptide was set at one . carbamidomethylation of cysteine was included as a fixed modification ; n - acetylation of the n terminus of the protein , oxidation of methionine , formation of pyro - glu from n - terminal glutamine , phosphorylation of serine , threonine , or tyrosine , and ubiquitination of lysine were all allowed as variable modifications . in searches of ltq - orbitrap xl or ltq - ft data , the mass tolerance was 30 ppm for precursor and 0.8 da for fragment ions . for qstar data , a precursor mass tolerance of 100 ppm and a fragment mass error tolerance of 0.2 da were allowed for the data search . the peak lists were searched against a subset of the uniprotkb database as of december 15 , 2009 , containing all entries for arabidopsis ( 53 624 entries searched ) . the false positive rate was estimated by searching the data using a concatenated database that contains the original arabidopsis uniprotkb database as well as a version of each original entry where the sequence was randomized . in all protein identifications , a minimal protein score of 22 , a peptide score of 15 , and a minimal discriminate score threshold of 0.0 were used for initial identification criteria . the maximum expectation value threshold ( number of different peptides with scores equivalent to or better than the result reported that are expected to occur in the database search by chance ) was set to 0.05 for accepting individual spectra and 0.01 for accepting individual proteins . when several accession numbers in the database corresponding to overlapping sequences of the same polypeptide were identified , the common gene locus and protein name were reported . only proteins with at least two peptides identified were further considered and reported . to assign the modification site for peptides containing post - translational modifications , the ms / ms spectrum was reinterpreted manually by matching the observed fragment ions to a theoretical fragmentation obtained using ms product ( proteinprospector ) . phosphatase treatment was performed as described elsewhere . in brief , 50 l of microsomal protein ( approximately 250 g ) in dige buffer was mixed with 5 l of 10% sds . then , 345 l of deionized water , 50 l of 20 mm mncl2 , and 50 l of 10 -protein phosphatase buffer were added sequentially , mixed , and incubated with 200 units of -protein phosphatase enzyme ( new england biolabs , ipswich , ma , usa ) overnight at 30 c . then , proteins were pelleted by adding five volumes of 0.1 m ammonium acetate in methanol and dissolved in dige buffer . the microsomal proteins were prepared as described above and were resuspended in the extraction buffer containing 0.1% ( w / v ) triton x-100 . after centrifugation at 20 000 g for 10 min , solubilized proteins were incubated with an anti - gfp antibody bound to protein a sepharose beads ( ge healthcare ) for 1 h. the beads were washed four times with the extraction buffer containing 0.1% triton x-100 and eluted with 2% sds . microsomal proteins and the immunoprecipitated proteins were resolved on 7.5% sds - page gel , transferred to a nitrocellulose membrane , and then stained with deep purple stain ( ge healthcare ) , as described . ubiquitination of phot1 was detected by western blot using an anti - ubiquitin antibody ( fk2 , mouse monoclonal , enzo life science , farmingdale , ny , usa ) , which detects both mono- and polyubiquitinylated proteins . blue light acts as an environmental cue to regulate plant growth and development , but its perception and transduction by plants remains poorly characterized . to understand better the early events of blue light signaling , the experimental protocol illustrated in figure 1 was employed to study the effect of blue light on the proteome of arabidopsis seedlings . here , we used 2d dige coupled with tandem mass spectrometry to identify early blue light - responsive proteins and protein modifications . four - day - old etiolated seedlings were irradiated with blue light at a fluence rate of 20 mol m s for 20 min , a total fluence that is known to saturate the phosphorylation of phot1 . because blue light - induced phototropism is initiated by membrane - associated receptors phot1 and phot2 , crude membrane proteins ( microsomal fraction ) from blue light - irradiated and unirradiated seedlings were compared by 2d dige . two rows of high - molecular - weight proteins ( approximately 120 kda ) showed clear mobility shifts in which the more acidic ones showed up only after irradiation and exhibited slightly lower electrophoretic mobility , whereas the more basic ones disappeared after irradiation ( figure 2a ) , which is the pattern that one would expect to see for light - induced protein phosphorylation . 2d dige analysis of the blue light response in the microsomal fraction of 4 day old etiolated arabidopsis seedlings . ( a , b ) col-0 seedlings were irradiated with blue light for 20 min , and microsomal proteins from both control ( unirradiated , proteins labeled with cy3 ) and irradiated seedlings ( proteins labeled with cy5 ) were analyzed by 2d dige . ( a ) superimposed 2d dige image of the upper - half of a gel . proteins ( in different modification isoforms ) induced by blue light treatment appear as red spots , and those decreased by the treatment appear green , whereas those remaining constant appear yellow . spots that were characterized by mass spectrometry are highlighted by arrows , and their identities are listed in table 1 . ( b ) zoomed - in 2d dige overlay image ( from a different gel than that in panel a ) showing the blue light - stimulated accumulation of web1 in the microsomal fraction . ( c ) identities of the row of phot1 spots were further confirmed by 2d dige analysis of the microsomal protein of the etiolated seedlings of the phot15 ( labeled with cy3 , green ) and gl ( its genetic background , labeled with cy5 , red ) mutants . the estimated ph ranges following ief are indicated above the gel images in figures 24 . in - gel trypsin digestion of an excised spot followed by liquid chromatography tandem mass spectrometry ( ms / ms ) identified these spots as phot1 ( figure 2a , arrow 1 , and table 1 ) , a result that was expected because phot1 has an apparent size of 120 kda . the identities of the row of phot1 spots from the unilluminated control seedlings were further confirmed by 2d dige comparison of phot15 with its genetic background , gl , as these spots disappeared in the phot15 mutant ( figure 2c ) . two other blue light - responsive protein spots with higher electrophoretic mobility were also identified as phot1 ( figure 2a , arrows 4 and 5 ) , which are likely the partial degradation products of phot1 . in addition to phot1 , a blue light - responsive protein was identified as web1 ( weak chloroplast movement under blue light 1 , figure 2 , arrows 2 and 3 ) , an acidic high - molecular - weight protein regulating the movement velocity of chloroplast photorelocation . web1 was previously shown to be predominantly in the soluble fraction of the proteome , with only a little found in the microsomal fractions . this is in agreement with its low abundance in the microsomal fraction in our study ( figure 2b ) . the accumulation of web1 protein in the microsomal fraction after blue light irradiation suggests that light induces web1 association with the cell membrane or some other cellular structure . as shown in figure 2a , proteins spots responsive to blue light were screened by decyder software and were further characterized by ms / ms . the spot volume ratios of blue light - irradiated to untreated ( positive numbers ) or untreated to irradiated ( negative numbers ) and the p values of the student s t - test of the comparison were calculated from three biological replicates . for the ms / ms identification , the number of unique peptides , percentage of sequence coverage , and e value ( best expectance value ) for each spot are listed . the two rows of phot1 spots extended from the basic to the acidic region in the gel shown in figure 2a . to determine whether all of these spots are the result of differential phosphorylation of phot1 , -protein phosphatase treatment , which hydrolyzes the phosphate groups from serine , threonine , tyrosine , and histidine residues , was performed on the microsomal proteins from both dark and blue light - irradiated seedlings . in both cases , phosphatase treatment shifted phot1 to the more basic regions and increased its electrophoretic mobility ( figure 3a , b ) . however , phot1 was still found as a string of spots in the 2-de gels after the phosphatase treatment , possibly the result of incomplete phosphatase digestion and/or post - translational modifications other than phosphorylation ( i.e. , any modifications affecting charge of the protein ) . phosphorylation is the major form of phot1 post - translational modification , as indicated by -phosphatase treatment . phosphatase treatment induced shifts of phot1 spots in microsomal protein from both blue light - irradiated ( a ) and control ( b ) seedlings . on the left , blue and blue/pp indicates the comparison of phosphatase - treated ( cy5 , red ) verse untreated ( cy3 , green ) microsomal proteins from blue light - irradiated seedlings by 2d dige and dark and dark/pp indicates the comparison of phosphatase treated ( cy5 , red ) verse untreated ( cy3 , green ) proteins from unirradiated seedlings . proteins that show up in the phosphatase - treated samples appear red , whereas those in the untreated samples are green . red arrows point to phot1 spots from samples of irradiated seedlings , green arrows point to those from the unirradiated samples , and white arrows point to those from phosphatase - treated proteins . salomon et al . found that the phosphorylation of phot1 in avena sativa was , in some way , hierarchical , with certain sites phosphorylated at low blue light fluences and other sites phosphorylated at higher blue light fluences . we applied 2d dige to study the time - dependent phot1 phosphorylation and dephosphorylation patterns in arabidopsis . as shown in figure 4a , a 2 min irradiation at a fluence rate of 20 mol m stimulated phot1 phosphorylation , as indicated by the acidic shift . a 5 min irradiation shifted phot1 further to the more acidic regions and appeared to saturate phot1 phosphorylation , as longer time treatment ( 20 and 60 min , respectively ) did not cause any further spot shifts in the acidic regions . in contrast , dephosphorylation of phot1 in a sequential order was observed when the saturation - illuminated ( 20 min ) seedlings were kept in the dark . after a 10 min recovery period in darkness , the charges in phot1 were shifted partially toward a more basic pi , as shown on the 2d dige gel , and after 60 min , there were no further shifts in electrophoretic mobility . time - dependent phot1 phosphorylation ( a ) and dephosphorylation ( b ) after irradiation of dark - grown seedlings . ( a ) etiolated col-0 seedlings were irradiated with blue light for the indicated times , and the microsomal proteins from irradiated ( red ) and unirradiated samples ( green ) were compared by 2d dige . shown ( b ) after saturating irradiation for 20 min , the etiolated seedlings were kept in the dark . the subsequent phot1 dephosphorylation was monitored by 2d dige , comparing the microsomal protein from the col-0 seedling collected at the time points indicated with that of the control samples ( collected immediately after irradiation ) . two previous studies identified eight different in vivo phot1 phosphorylation sites , but the large number of phot1 spots with distinct pi values ( figures 24 ) that were shifted by protein phosphatase treatment ( figure 3 ) suggested the existence of additional phot1 phosphorylation sites . to identify other possible phot1 phosphorylation sites , an anti - gfp antibody was used to immunoprecipitate phot1gfp from the microsomal proteins of both irradiated and unirradiated arabidopsis seedlings expressing phot1gfp , and the immunoprecipitates were analyzed by tandem mass spectrometry after separation by sds - page . in the phot1gfp proteins from dark - grown seedlings , three phosphorylated ser sites were observed ( supporting information figure 1 ) , with all of them located in the n - terminus ( figure 5 ) . in the illuminated samples , 13 phosphorylation sites were identified ( figure 5 and supporting information figure 1 ) , which were localized both to the n - terminus and the hinge region between lov1 and lov2 ( figure 5 ) , including two sites ( s58 and s170 ) that were also detected in the dark sample . a representative annotated tandem mass spectrum of phot1 phosphopeptides is shown in figure 6 ( for the identity of the phosphorylation sites , see table 2 ) . in all because six of the sites ( s58 , s170 , s185 , s350 , s376 , and s410 ) were reported to be phosphrylated in vivo either in the dark or in illuminated samples , we report here eight additional in vivo phosphorylated sites ( indicated in red in figure 5 ) phosphorylated ser / thr sites identified from blue light - treated tissues are shown above the protein box , whereas those identified from the dark tissues are shown below the protein box . ser / thr sites in black were identified both in this study and in previous studies , those in blue were not identified in this study but were reported previously , and those in red are new sites identified in this study . phosphorylated s170 was identified from phot1 protein in both dark and irradiated seedlings in this study , but it was identified only from phot1 protein in dark seedlings in a previous study . in summary , eight new unique sites ( s12 , s92 , s141 , t144 , t360 , s406 , s442 , and s450 ) were identified in this study . representative cid ms / ms spectrum of phot1 obtained from a precursor ion with a m / z value of 910.7604 , corresponding to a phosphorylated peptide spanning the residues s75k98 of phot1 ( theoretical monoisotopic mass value , 910.7576 ; error in the mass observed in the precursor ion , 3.1 ppm ) . phosphorylated serine is shown in the sequence as s. the observed b and y product ion peaks are labeled accordingly , with the subscripts denoting their position in the identified peptide . intense ions corresponding to neutral losses from the labeled sequence ions are indicated by * . in the peptide sequence , mass spectra for peptides in this table are provided in figure 6 and in supporting information figure l. s * and t * represent phosphorylation sites . interestingly , sequence alignment showed that s185 ( in phot1 ) or s121 ( in phot2 ) , located in a highly conserved amino acid region just before lov1 domain of phot1/2 , is the only known conserved site phosphorylated in both proteins in vivo ( supporting information figure 2 ) . ubiquitination of phot1 has been shown to be involved in phototropic responses , but the ubiquitination site(s ) of phot1 have not yet been characterized . key features for detection of potential ubiquitin attachment sites on tryptic peptides are the missed cleavage of the modified lysine together with a shift of 114 da ( a diglycine moiety , gg ) in both the mass of the precursor ion and the masses of sequence fragment ions in the ms / ms spectrum that would contain the ubiquitin - modified site of the peptide . these gg remnants result from the cleavage by trypsin of the original ubiquitin in the c - terminal side or r on its c - terminal ggr sequence . accordingly , a unique ubiquitination site ( k526 ) , localized in the lov2 region , was observed in the spectrum of the peptide f514k527 of phot1gfp from blue light - irradiated seedlings ( figure 7 ) , but it was not observed in the dark controls ( data not shown ) . to document that ubiquitination of phot1 was regulated by blue light irradiation , phot1gfp proteins immunoprecipitated by an anti - gfp antibody were immunoblotted with an anti - ubiquitin fk2 antibody that detects both mono- and polyubiquitinylated proteins . immunoblot assays show that phot1 ubiquitination was enhanced after blue light irradiation , whereas it was reduced during recovery in the dark , in a pattern similar to that of phot1 phosphorylation ( figure 8) . cid ms / ms spectrum obtained from a precursor ion with a m / z value of 845.9529 , corresponding to a ubiquitinated peptide spanning residues f514k527 of phot1 ( theoretical monoisotopic mass value , 845.9542 ; error in the mass observed in the precursor ion , 1.4 ppm ) . ubiquitinated lysine is shown in the sequence as k , as it is labeled by the digly tryptic remnant of ubiquitin . thereafter , one - third of the samples were collected immediately under dim safe light ( dark ) , another third were irradiated with blue light for 20 min ( blue ) , and the remaining seedlings were incubated in the dark for 60 min after 20 min of blue light irradiation ( recovery ) . microsomal proteins were extracted from these seedlings and immunoprecipitated with an anti - gfp antibody . the microsomal proteins ( input ) and the immunoprecipitates ( ip ; anti - gfp ) were resolved by a 7.5% sds - page gel , transferred to a nitrocellulose membrane , stained by deep purple fluorescent stain ( a ) , and detected by an anti - ubiquitin antibody ( b ) . in panel a , phot1gfp with lower and higher electrophoretic mobilities is marked with stars . in panel b , ubiquitination of phot1 crop yield is , to a large extent , dependent on the photosynthetic activity of the green leaves of the developing plant . recent studies have shown that the photosynthetic efficiency of these organs is optimized via a number of blue light / uv - a - mediated responses , such as phototropism in the upper region of the plant , chloroplast movement , stomatal opening , palisade cell development , and leaf flattening . in the model organism a. thaliana , all of these physiological processes are mediated by phototropins ( phot1 and phot2 ) , as reviewed by christie et al . however , the signaling cascade that leads from the photoexcitation of phot1 ( or phot2 ) to the corresponding physiological response has not yet been elucidated in detail . the aim of this study was to explore this signaling pathway in arabidopsis further . we detected the phosphorylation of phot1 and accumulation of web1 in the membrane - associated protein fraction by 2d dige ( figures 24 ) , and we identified a large number of phot1 ser / thr sites phosphorylated in vivo ( figure 5 ) as well as a phot1 ubiquitination site , which was identified by mass spectrometry analysis of phot1gfp co - immunoprecipitates ( figure 7 ) . these results further revealed the complexity of phot1 post - translational modifications . since phototropins were first found to be membrane - associated proteins that are phosphorylated upon blue light irradiation in etiolated seedlings of pea ( pisum sativum ) , much progress has been made in characterizing the phosphorylation of phototropins . salomon et al . identified eight ser residues in oat phot1a in vitro that are phosphorylated , which are hierarchically located either in the n - terminus or hinge 1 region . sullivan et al . identified four phosphorylation sites in vivo in arabidopsis phot1 : two in the n - terminus and two in the hinge 1 region . inoue et al . identified eight ser / thr residues in arabidopsis phot1 , including one in the kinase domain and one in the c - terminus ( that we did not detect ) in addition to six in the n - terminus or hinge 1 region . interestingly , all the four sites identified by sullivan et al . were observed by inoue et al . however , the large number of individual protein spots ( over 50 ) from the two rows of phot1 spanning from the basic to the acidic regions detected in our 2d dige gels ( figure 2a ) indicates the greater complexity of phot1 phosphorylation . our study identified 14 discrete ser / thr sites phosphorylated in vivo , which included six sites identified by sullivan et al . and inoue et al . in addition , we identified eight additional ser / thr sites phosphorylated in vivo from unirradiated and blue light - exposed samples . a string of parallel phot1 spots was detected in the unirradiated etiolated samples ( figure 2a , c ) , and they were shifted to more basic regions after phosphatase treatment , indicating that some sites of phot1 were phosphorylated even in the dark . in this study , we identified three such phosphoserine residues from phot1 in seedlings without blue light treatment ( figure 5 ) . despite the large number of phosphorylation sites identified for phototropins in this and other studies , kinoshita et al . reported the binding of a 14 - 3 - 3 protein to s358 in vicia faba phot1a and s344 in v. faba phot1b and proposed that the binding is likely a key step in the phototropin - mediated stomatal opening response . report the binding of a 14 - 3 - 3 protein to s350 and s376 of arabidopsis phot1 ; because s376 corresponds to s344 in v. faba phot1b , it could possibly serve the same role . inoue et al . showed that autophosphorylation of s851 in the kinase activation loop was essential for a whole series of phot1-mediated responses in arabidopsis and proposed a possible secondary role for s849 . finally , tseng et al . demonstrated that s747 in arabidopsis phot2 bound the isoform of a 14 - 3 - 3 protein in a yeast two - hybrid screen and that mutating that serine to alanine blocked phot2-mediated stomatal opening . the mutation failed to have an impact on phototropism , however , indicating a high level of specificity . sorting out the roles of the many phototropin phosphorylations remains a daunting task . a string of protein spots was observed in the phot1 region in the microsomal protein samples from either the unirradiated or irradiated seedlings , even after overnight phosphatase treatment of the microsomal proteins , suggesting additional post - translational modifications other than phosphorylation and/or incomplete phosphatase treatment . in additional to protein phosphorylation , ubiquitination is another important post - translational modification that regulates protein activity , including protein degradation , membrane protein endocytosis , and subcellular protein trafficking . showed that phot1 could be mono- , multi- , or polyubiquitinated , depending on light intensity , and phot1-interacting protein nonphototropic hypocotyl 3 ( nph3 ) functions as a substrate adaptor in an e3 cullin3-based ubiquitin ligase . they also showed that their high - intensity light treatment failed to induce any detectable ubiquitination in vivo in an nph3 mutant , indicating that nph3 is involved in the ubiquitination that we detected . in addition , we found that ubiquitination of phot1 followed a pattern similar to that for phosphorylation : both increased after blue light exposure but subsequently decreased during the recovery stage following a saturating light exposure . it seems likely that ubiquitination in addition to phosphorylation could account for the increase in mass that we detected for phot1 on illumination in vivo . lys526 is very close to the attachment of the j- helix to the lov2 domain , so it is reasonable to expect that it could affect lov domain unfolding following photoexcitation or folding during dark recovery . in this manner a tryptophan , w491 , is also extremely close to lys526 , and hoersch et al . presented evidence that this tryptophan is moved into a more hydrophilic environment upon lov domain photoexcitation , indicative of the protein conformational changes in this region of the molecule . because they are unstable , of low abundance , and often in low stoichiometry , phosphopeptides are hard to detect by mass spectrometry alone , and immunoprecipitation or other methods are often used to enrich specific proteins to identify phosphopeptides . even so , only a small number of phosphopeptides has been previously identified for phot1 . however , 2d dige , which separates two or three samples in the same gel , can detect slight differences in pi as a result of a change in phosphorylation at any residue of the protein . therefore , the shifted spots in 2d dige provide a more accurate estimate of protein phosphorylation sites . as shown before , 2-de gels resolved over 20 spots of the brassinosteroid - signaling transcription factor bzr1 , which is consistent with the number of phosphorylation sites predicted on the basis of the consensus substrate sequence for the bin2/gsk3 kinase . only with the combination of protein fractionation and enrichment , tandem mass spectrometry , and 2d dige can a more accurate picture of specific protein phosphorylation and dephosphorylation be elucidated . our study and previous studies showed that more than 16 phosphorylation sites and a number of other post - translationally modified residues affecting charge are likely present for phot1 . further studies will be required to characterize the biochemical complexity of the phot1 protein and its physiological implications . except for the phosphorylation sites in the kinase domain , the function of other phosphorylation sites is not known for phototropin - mediated signaling , but it might be expected to play some role in one or more blue light - induced physiological responses .

plants adapt to environmental light conditions by photoreceptor - mediated physiological responses , but the mechanism by which photoreceptors perceive and transduce the signals is still unresolved . here , we used 2d difference gel electrophoresis ( 2d dige ) and mass spectrometry to characterize early molecular events induced by short blue light exposures in etiolated arabidopsis seedlings . we observed the phosphorylation of phototropin 1 ( phot1 ) and accumulation of weak chloroplast movement under blue light 1 ( web1 ) in the membrane fraction after blue light irradiation . over 50 spots could be observed for the two rows of phot1 spots in the 2-de gels , and eight novel phosphorylated ser / thr sites were identified in the n - terminus and hinge 1 regions of phot1 in vivo . blue light caused ubiquitination of phot1 , and k526 of phot1 was identified as a putative ubiquitination site . our study indicates that post - translational modification of phot1 is more complex than previously reported .

Introduction Materials and Methods Results Discussion

PMC4076864

circadian rhythms are discernible in physiological and biochemical properties of the human body , such as core body temperature and corticosteroid and melatonin levels . this time keeping system , termed the endogenous clock , has evolved into a free - running entity that produces circadian rhythms even when time zeitgebers are absent . an average human suprachiasmatic nuclei ( scn ) , a pacemaker which controls the mammalian timing system , runs with a period slightly exceeding 24 hours and the scn consists of neuron clusters whose electrical potential frequency fluctuates spontaneously with an approximate 24-hour periodicity . circadian clock provides the optimization of metabolism and energy utilization for sustaining life processes in the organism . it is clear that daily food intake , metabolism and detoxification as well as several physiological processes are regulated by the circadian rhythm . chronopharmacologic phenomena can be viewed as resulting from an adaptation of the organism to cyclic environmental changes in 24 hours and the toxicity of xenobiotics is modified by the chronicity of cellular activity of organs . in toxicology , animal housing of rodents are performed according to 12 hours dark and light cycles to control the secretions of several hormones ; however , applications in clinic according to the circadian rhythm have only gained attention in the last decades . we know that the circadian pharmacokinetics and particularly pharmacodynamics that modulate drug effectiveness and toxicity are manifestations of the circadian regulation of xenobiotic detoxification and could have important clinical applications since they allow treatment modification in order to increase efficacy and safety of a certain drug or decrease side effects . however , studies omitting the timing of dosing may be the partial cause of the reports that found so many substances carcinogenic . different cells of different functional organs have different periodicities and their functions are influenced by circadian changes at varying levels . moreover , the gender , age , physical activity , disease states and phenotypic properties of the individual have a big impact on these functions . concerning the effects of circadian clock , successful applications of drug therapy in asthma , allergic diseases , psychotic states , hypertension , coronary heart disease and diabetes were achieved . in this review , we will focus on mammalian circadian pharmacodynamics and discuss new chronotherapy approaches . moreover , we will try to highlight the chronopharmacodynamics of cardiovascular drugs , anti - cancer drugs , analgesics and non - steroidal anti - inflammatory drugs and will give some practical concerns for clinical pharmacists and pharmacy practitioners . pharmacodynamics is the study of the biochemical and physiological effects of drugs on the body as well as the mechanisms of drug action and the relationship between drug concentration and its effect . one particular example may be drug - receptor interactions . biological rhythms at the cellular and subcellular levels can provoke significant dosing - time differences in the pharmacodynamics of medications that are unrelated to their pharmacokinetics . firstly , this term was used to describe rhythmic and temporally predictable alterations in the susceptibility or the sensitivity of a target biological system to a drug , which can not be expounded by chronokinetic changes . afterwards , considering many experimental findings chronesthesy was considered as the chronopharmacologic counterpart of the pharmacodynamic concept . chronopharmacodynamics basically endeavors with mechanisms of time - related variation in effects and metabolism of drugs in healthy organisms . however , the term chronesthesy can be used instead of chronopharmacodynamics which is a long and difficult word . rhythms in receptor number or conformation , secondary messengers , metabolic pathways , and/or free - to - bound fraction of medications help explain this phenomenon . here there is evidence in literature that circadian clock has impact on the pharmacodynamics of several cardiovascular drugs . the cardiovascular drugs that have evidence for the impact of circadian rhythm on their pharmacodynamics beta - receptor blocking drugs ( -blockers ) consist of an important group of cardio - active drugs . these medications still have great therapeutic value in the treatment of cardiovascular disorders like coronary heart disease , hypertension and arrhythmias . different -blockers vary not only in their specific effects like receptor affinity , receptor selectivity , intrinsic sympathomimetic activity , and in their nonspecific effects related to the lipophilicity of the drug , but also in their biotransformation . lipophilic -blockers are usually subject to hepatic biotransformation whereas hydrophilic ones are mainly eliminated by renal excretion . various -blockers have been studied in several animal experiments and in volunteers for their chronopharmacodynamic properties . the peak concentration of propanolol was achieved in the application between 8 a.m. and 2 p.m .. however , when applied on 2 a.m. , the heart rate can be slightly changed in the following 6 hours . therefore , it can be stated that sympathetic tonus , which is demonstrated by the rhythm in plasma noradrenaline and camp levels , affects the pharmacodynamics of the particular drug and it can be concluded that propanolol should be applied in hours when sympathetic tonus is high . anti - anginal drugs such as nitrates favorably shift the ratio of myocardial oxygen demand and supply , by relieving chest pain and reducing the duration and frequency of acute ischemic events . isosorbidedinitrate , an important nitrate derivative , shows its highest therapeutic effect around 2 a.m. when it highly induces decrease in blood pressure and increase in heart rate . in contrast , at 8 a.m. isosorbitedinitrate did not significantly increase reflexly induced tachycardia during orthostatis . these findings are also in concert with the data that a maximum orthostatic liability is observed around 3 a.m. calcium channel blockers are used in the treatment of coronary heart disease , myocardial infarct , cerebrovascular diseases and hypertension for several years . verapamil and diltiazem have a more prominent cardiac effect , whereas dihydropyridine type blockers such as nifedipine have a more dominant vasodilator effect . several chronopharmacologic studies were performed on various calcium channel blockers and in general their blood pressure lowering effect was found to be higher in daytime than night time and the circadian clock dependent effects of these drugs show similar pattern as -blockers . in clinical practice , doctors and other health care personnel do not pay adequate importance for the time of administration of anti - cancer therapy . besides , the timing for anti - cancer drug administration is rarely indicated in study protocols or seldom reported in the reports from clinical trials . as a consequence inter - individual and intra - individual variations can be observed . for the convenience of hospital staff , most intravenous chemotherapy is being applied during daytime and oral drugs are usually administered once daily , preferentially administered in morning , without putting forward a rationale for this time of dosing . this conventional approach can not be used for some of the drugs as this lack of attention to the timing of therapy supposes that biological parameters are either constant throughout the 24 hours , or that their variations are unpredictable and/or stochastic . however , the timing of therapy must be based on data on appropriate circadian rhythms obtained from rodent and human chronotherapy studies in order to rationalize the administration time for each drug and optimize its therapeutic index . for anti - cancer drugs used in infusion form , in order to define the drug regimen , dose administered per unit ( usually body surface area or body weight ) , duration of infusion and frequency of administration are the parameters commonly used . if the cancer drug will be infused for a short time , chronotherapy studies can be performed with a timed bolus or a short infusion where the timing of administration ( start , peak and stop times ) are stipulated . on the other hand , a constant - rate infusion over 24 hours , or integral multiples of this span , therefore , this procedure should be used as a control administration schedule for studies of cancer chronotherapy for drugs whose pharmacologic properties enable long term infusion . the dissimilarities between the conventional and the chrono - modulated applications are not limited to the time of administration , but include differences in delivery profile , infusion duration and drug sequence . a new anti - cancer delivery system named as chrono - modulated delivery introduces another infusion parameter , the time of peak - flow rate . in this form of application it is rather semi - sinusoidal , with an increasing flow rate , a peak administration rate at a time specification and a gradual symmetric decrease in flow rate . the chrono - modulated application profile is particularly convenient for drugs with a short half - life where a relatively long duration of administration is choosen . here we can give the examples for the change in the pharmacodynamics of some anti - cancer drugs : dna synthesis in the main target tissues of 5-fluorouracil ( 5-fu)-induced toxicity ( e.g. , bone marrow , skin , and oral and rectal mucosa ) is lowest during the night and highest during daytime . therefore , at night when the whole - body clearance of 5-fu is increased , the proportion of healthy cells potentially damaged by 5-fu is decreased . whole - body pharmacodynamics of 5-fu , therefore displays variation along the circadian time scale , with a synchronous phase between different target tissues . the anabolic enzymes ( orotatephosphoribosyltransferase , uridinephosphorylase , and deoxythymidine kinase ) that produce cytotoxic forms of 5-fu have their highest activity during the dark span of rats or mice , when 5-fu is most toxic to healthy tissues . the activity of the thymidylatesynthetase , which is the target enzyme of 5-fu has also been studied at the cellular level in the oral mucosa cells of 6 healthy volunteers . the activity of this particular enzyme showed a circadian rhythm with a trough between midnight and 4 a.m. therefore , the molecular target of 5-fu is less active at night . this results in a cellular chronopharmacodynamic pattern of this drug consistent with its lower cytotoxicity to the oral mucosa during the night . the circadian profiles of whole - body and cellular chronopharmacokinetics and chronopharmacodynamics in humans would therefore predict a better tolerability of healthy tissues for a nightly administration of 5-fu . the anti - tumor effects of interferon- ( ifn- ) in mice are more efficient during the early rest phase than during the early active phase . the dosing schedule - dependent effect of ifn- is also closely related to that of ifn receptors and interferon - stimulated gene factor ( isgf ) expression in tumor cells or lymphocytes . tyrosine kinase is an enzyme that transfers a phosphate group from atp to a protein in the cell . imatinib mesylate is a molecule that inhibits the function of various receptors with tyrosine kinase activity . in mice , the influence of dosing time on the ability of imatinib to inhibit tumor growth has been investigated . it was observed that the growth of tumor cells implanted in mice was more severely inhibited by the administration of imatinib during the early rest phase than during the early active phase . the dosing time - dependency of anti - tumor effects is parallel to that of the imatinib - induced antiangiogenic effect . therefore , the potent therapeutic efficacy of the drug could be expected by optimizing the dosing schedule . both in rodents and in humans , biological rhythms in pain sensitivity have been studied extensively . morphine shows a broad range of other pharmacological effects in addition to its potent analgesic properties . in humans , reduction in gastrointestinal motility , sedation , inhibition of the micturition reflex , and miosis were observed . in rodents , though some of the pharmacologic effects were similar ; different reactions were also found , like mydriasis as an opposite reaction . pharmacological tolerance to the analgesic effect of morphine was observed in several studies after chronic administration of morphine in both experimental and in humans . however , the clinical tolerance in humans develops more slowly . however , chronopharmacological studies on the development of tolerance to the analgesic effect induced by morphine are very rare . this should be an important research field for scientists as such opioids are important pain killers , especially for the end - stage of cancer . among few studies performed on morphine chronopharmacodynamics , yoshida et al . , ( 2003 ) used hot - plate method induce pain in mice and the researchers detected the chronopharmacodynamic response towards morphine by using this model . the researchers found that there was a significant 24 hours rhythm in the latency of thermal response after morphine injection with a trough at the light phase and a peak at the dark phase . especially , at the dark phase , the time spent on the hot - plate after morphine injection was significantly longer compared with non - drugged state . the rhythmic pattern of analgesic effect induced by morphine was similar to that of the sensitivity of mice to painful stimuli in non - drugged state . the results of that study were consistent with the previous findings performed by naloxane and morphine . chronopharmacological studies were also performed on the heat - pain reducing effects of fentanyl in humans . a peak in pain relief occurred late in the afternoon ( 5.30 p.m. ) and a trough in the early morning hours ( 5.30 a.m. ) . on the other hand , the analgesic effects of two other opioids [ dihydrocodeine ( dhc ) and tramadol ] the results indicated that the painful intensity of the chemical stimuli strongly increased during evening sessions . in addition , both dhc and tramadol exerted stronger analgesic effects when administered in the evening . the kaolin - induced pain mouse model seems to be useful for the chronopharmacodynamic evaluation of analgesic agents . in a study using this model , chronopharmacodynamic effect of indomethacin , a nsaid drug , indomethacin was adminstered orally to mice at 2 a.m. , 8 a.m. , 2 p.m. , or 8 p.m. , and the suppressive effect on kaolin - induced writhing was determined after each timed dosing . after dosing at 8 a.m. , indomethacin remarkably decreased the number of writhes during the critical span of 2 - 6 p.m. , the time when writhing reaction was greatest during the 24 hours , while the suppressive effect of the medicine after dosing at the other clock times was relatively small . these findings suggest the analgesic effect of indomethacin in mice with the kaolin - induced writhing is greater after dosing in the early resting period , which is similar to that reported in patients with nocturnal pain . desired effects or side effects of medications are dependent not only on the physico - chemical properties of the compound , but also on its dosage , its pharmacokinetics , and particularly on its pharmacodynamics . as pharmacodynamics is affected by circadian variability , circadian rhythm should be taken into account before the administration of a drug in order to prevent the temporal variations in the mode of action . therefore , clinical pharmacists and pharmacy practitioners should take into account that the time of administration might be very important in eliminating the risk of toxicity of certain medications , particularly cardio - active , anti - cancer and nsaid drugs . for -blockers , particularly for propanolol , the time of administration should be between 8 a.m. and 2 p.m. as peak concentration is achieved when the sympathetic tonus is high . similarly , the blood pressure lowering effect of calcium channel blockers was found to be higher in daytime than night time . besides , as hypertension , heart attack and stroke mostly occurs in morning time , daytime administration of these drugs gives opportunity to prevent diseases of cardio - vascular origin . for anti - cancer drugs , rather than choosing daytime administration for the convenience of the health care personnel , a valid application procedure should be administered . as the chronopharmacodynamic properties of each drug as well as each pharmaceutical form ( infusion , oral ) might differ from each other , the right administration time should be chosen according to studies performed on each particular medication . besides , the manufacturers should be forced to include the time of administration to the package inserts of these drugs . nsaids comprise a very important group of medications , used in a variety of diseases . these drugs should be administered at morning time when the pain is at its highest , especially in rheumatoid arthritis . morning time administration usually provides a day - long reduction in pain and an increase in life quality . pharmacodynamics is the study of the biochemical and physiological effects of drugs on the body as well as the mechanisms of drug action and the relationship between drug concentration and its effect . . biological rhythms at the cellular and subcellular levels can provoke significant dosing - time differences in the pharmacodynamics of medications that are unrelated to their pharmacokinetics . firstly , this term was used to describe rhythmic and temporally predictable alterations in the susceptibility or the sensitivity of a target biological system to a drug , which can not be expounded by chronokinetic changes . afterwards , considering many experimental findings chronesthesy was considered as the chronopharmacologic counterpart of the pharmacodynamic concept . chronopharmacodynamics basically endeavors with mechanisms of time - related variation in effects and metabolism of drugs in healthy organisms . however , the term chronesthesy can be used instead of chronopharmacodynamics which is a long and difficult word . rhythms in receptor number or conformation , secondary messengers , metabolic pathways , and/or free - to - bound fraction of medications help explain this phenomenon . here there is evidence in literature that circadian clock has impact on the pharmacodynamics of several cardiovascular drugs . the cardiovascular drugs that have evidence for the impact of circadian rhythm on their pharmacodynamics beta - receptor blocking drugs ( -blockers ) consist of an important group of cardio - active drugs . these medications still have great therapeutic value in the treatment of cardiovascular disorders like coronary heart disease , hypertension and arrhythmias . different -blockers vary not only in their specific effects like receptor affinity , receptor selectivity , intrinsic sympathomimetic activity , and in their nonspecific effects related to the lipophilicity of the drug , but also in their biotransformation . lipophilic -blockers are usually subject to hepatic biotransformation whereas hydrophilic ones are mainly eliminated by renal excretion . various -blockers have been studied in several animal experiments and in volunteers for their chronopharmacodynamic properties . the peak concentration of propanolol was achieved in the application between 8 a.m. and 2 p.m .. however , when applied on 2 a.m. , the heart rate can be slightly changed in the following 6 hours . therefore , it can be stated that sympathetic tonus , which is demonstrated by the rhythm in plasma noradrenaline and camp levels , affects the pharmacodynamics of the particular drug and it can be concluded that propanolol should be applied in hours when sympathetic tonus is high . anti - anginal drugs such as nitrates favorably shift the ratio of myocardial oxygen demand and supply , by relieving chest pain and reducing the duration and frequency of acute ischemic events . isosorbidedinitrate , an important nitrate derivative , shows its highest therapeutic effect around 2 a.m. when it highly induces decrease in blood pressure and increase in heart rate . in contrast , at 8 a.m. isosorbitedinitrate did not significantly increase reflexly induced tachycardia during orthostatis . these findings are also in concert with the data that a maximum orthostatic liability is observed around 3 a.m. calcium channel blockers are used in the treatment of coronary heart disease , myocardial infarct , cerebrovascular diseases and hypertension for several years . verapamil and diltiazem have a more prominent cardiac effect , whereas dihydropyridine type blockers such as nifedipine have a more dominant vasodilator effect . several chronopharmacologic studies were performed on various calcium channel blockers and in general their blood pressure lowering effect was found to be higher in daytime than night time and the circadian clock dependent effects of these drugs show similar pattern as -blockers . in clinical practice , doctors and other health care personnel do not pay adequate importance for the time of administration of anti - cancer therapy . besides , the timing for anti - cancer drug administration is rarely indicated in study protocols or seldom reported in the reports from clinical trials . as a consequence inter - individual and intra - individual variations can be observed . for the convenience of hospital staff , most intravenous chemotherapy is being applied during daytime and oral drugs are usually administered once daily , preferentially administered in morning , without putting forward a rationale for this time of dosing . this conventional approach can not be used for some of the drugs as this lack of attention to the timing of therapy supposes that biological parameters are either constant throughout the 24 hours , or that their variations are unpredictable and/or stochastic . however , the timing of therapy must be based on data on appropriate circadian rhythms obtained from rodent and human chronotherapy studies in order to rationalize the administration time for each drug and optimize its therapeutic index . for anti - cancer drugs used in infusion form , in order to define the drug regimen , dose administered per unit ( usually body surface area or body weight ) , duration of infusion and frequency of administration are the parameters commonly used . if the cancer drug will be infused for a short time , chronotherapy studies can be performed with a timed bolus or a short infusion where the timing of administration ( start , peak and stop times ) are stipulated . on the other hand , a constant - rate infusion over 24 hours , or integral multiples of this span , therefore , this procedure should be used as a control administration schedule for studies of cancer chronotherapy for drugs whose pharmacologic properties enable long term infusion . the dissimilarities between the conventional and the chrono - modulated applications are not limited to the time of administration , but include differences in delivery profile , infusion duration and drug sequence . a new anti - cancer delivery system named as chrono - modulated delivery introduces another infusion parameter , the time of peak - flow rate . in this form of application it is rather semi - sinusoidal , with an increasing flow rate , a peak administration rate at a time specification and a gradual symmetric decrease in flow rate . the chrono - modulated application profile is particularly convenient for drugs with a short half - life where a relatively long duration of administration is choosen . here we can give the examples for the change in the pharmacodynamics of some anti - cancer drugs : dna synthesis in the main target tissues of 5-fluorouracil ( 5-fu)-induced toxicity ( e.g. , bone marrow , skin , and oral and rectal mucosa ) is lowest during the night and highest during daytime . therefore , at night when the whole - body clearance of 5-fu is increased , the proportion of healthy cells potentially damaged by 5-fu is decreased . whole - body pharmacodynamics of 5-fu , therefore displays variation along the circadian time scale , with a synchronous phase between different target tissues . the anabolic enzymes ( orotatephosphoribosyltransferase , uridinephosphorylase , and deoxythymidine kinase ) that produce cytotoxic forms of 5-fu have their highest activity during the dark span of rats or mice , when 5-fu is most toxic to healthy tissues . the activity of the thymidylatesynthetase , which is the target enzyme of 5-fu has also been studied at the cellular level in the oral mucosa cells of 6 healthy volunteers . the activity of this particular enzyme showed a circadian rhythm with a trough between midnight and 4 a.m. therefore , the molecular target of 5-fu is less active at night . this results in a cellular chronopharmacodynamic pattern of this drug consistent with its lower cytotoxicity to the oral mucosa during the night . the circadian profiles of whole - body and cellular chronopharmacokinetics and chronopharmacodynamics in humans would therefore predict a better tolerability of healthy tissues for a nightly administration of 5-fu . the anti - tumor effects of interferon- ( ifn- ) in mice are more efficient during the early rest phase than during the early active phase . the dosing schedule - dependent effect of ifn- is also closely related to that of ifn receptors and interferon - stimulated gene factor ( isgf ) expression in tumor cells or lymphocytes . tyrosine kinase is an enzyme that transfers a phosphate group from atp to a protein in the cell . imatinib mesylate is a molecule that inhibits the function of various receptors with tyrosine kinase activity . in mice , the influence of dosing time on the ability of imatinib to inhibit tumor growth has been investigated . it was observed that the growth of tumor cells implanted in mice was more severely inhibited by the administration of imatinib during the early rest phase than during the early active phase . the dosing time - dependency of anti - tumor effects is parallel to that of the imatinib - induced antiangiogenic effect . therefore , the potent therapeutic efficacy of the drug could be expected by optimizing the dosing schedule . both in rodents and in humans , biological rhythms in pain sensitivity have been studied extensively . morphine shows a broad range of other pharmacological effects in addition to its potent analgesic properties . in humans , reduction in gastrointestinal motility , sedation , inhibition of the micturition reflex , and miosis were observed . in rodents , though some of the pharmacologic effects were similar ; different reactions were also found , like mydriasis as an opposite reaction . pharmacological tolerance to the analgesic effect of morphine was observed in several studies after chronic administration of morphine in both experimental and in humans . however , the clinical tolerance in humans develops more slowly . however , chronopharmacological studies on the development of tolerance to the analgesic effect induced by morphine are very rare . this should be an important research field for scientists as such opioids are important pain killers , especially for the end - stage of cancer . among few studies performed on morphine chronopharmacodynamics , yoshida et al . , ( 2003 ) used hot - plate method induce pain in mice and the researchers detected the chronopharmacodynamic response towards morphine by using this model . the researchers found that there was a significant 24 hours rhythm in the latency of thermal response after morphine injection with a trough at the light phase and a peak at the dark phase . especially , at the dark phase , the time spent on the hot - plate after morphine injection was significantly longer compared with non - drugged state . the rhythmic pattern of analgesic effect induced by morphine was similar to that of the sensitivity of mice to painful stimuli in non - drugged state . the results of that study were consistent with the previous findings performed by naloxane and morphine . chronopharmacological studies were also performed on the heat - pain reducing effects of fentanyl in humans . a peak in pain relief occurred late in the afternoon ( 5.30 p.m. ) and a trough in the early morning hours ( 5.30 a.m. ) . on the other hand , the analgesic effects of two other opioids [ dihydrocodeine ( dhc ) and tramadol ] were investigated in humans . the results indicated that the painful intensity of the chemical stimuli strongly increased during evening sessions . in addition , both dhc and tramadol exerted stronger analgesic effects when administered in the evening . the kaolin - induced pain mouse model seems to be useful for the chronopharmacodynamic evaluation of analgesic agents . in a study using this model , chronopharmacodynamic effect of indomethacin , a nsaid drug , indomethacin was adminstered orally to mice at 2 a.m. , 8 a.m. , 2 p.m. , or 8 p.m. , and the suppressive effect on kaolin - induced writhing was determined after each timed dosing . after dosing at 8 a.m. , indomethacin remarkably decreased the number of writhes during the critical span of 2 - 6 p.m. , the time when writhing reaction was greatest during the 24 hours , while the suppressive effect of the medicine after dosing at the other clock times was relatively small . these findings suggest the analgesic effect of indomethacin in mice with the kaolin - induced writhing is greater after dosing in the early resting period , which is similar to that reported in patients with nocturnal pain . desired effects or side effects of medications are dependent not only on the physico - chemical properties of the compound , but also on its dosage , its pharmacokinetics , and particularly on its pharmacodynamics . as pharmacodynamics is affected by circadian variability , circadian rhythm should be taken into account before the administration of a drug in order to prevent the temporal variations in the mode of action . therefore , clinical pharmacists and pharmacy practitioners should take into account that the time of administration might be very important in eliminating the risk of toxicity of certain medications , particularly cardio - active , anti - cancer and nsaid drugs . for -blockers , particularly for propanolol , the time of administration should be between 8 a.m. and 2 p.m. as peak concentration is achieved when the sympathetic tonus is high . similarly , the blood pressure lowering effect of calcium channel blockers was found to be higher in daytime than night time . besides , as hypertension , heart attack and stroke mostly occurs in morning time , daytime administration of these drugs gives opportunity to prevent diseases of cardio - vascular origin . for anti - cancer drugs , rather than choosing daytime administration for the convenience of the health care personnel , a valid application procedure should be administered . as the chronopharmacodynamic properties of each drug as well as each pharmaceutical form ( infusion , oral ) might differ from each other , the right administration time should be chosen according to studies performed on each particular medication . besides , the manufacturers should be forced to include the time of administration to the package inserts of these drugs . nsaids comprise a very important group of medications , used in a variety of diseases . these drugs should be administered at morning time when the pain is at its highest , especially in rheumatoid arthritis . morning time administration usually provides a day - long reduction in pain and an increase in life quality . circadian rhythms are endogenous in nature and have been well - documented for eukaryotic organisms . the important feature of endogenous biological rhythms is their anticipatory character . rhythmicity inherent to all living systems provides an easier adaptation and better survival under different environmental conditions during the 24 hours cycle as well as during changing seasons . considering this knowledge , it is conceivable that not only the right amount of the right substance must be at the right place , but also this must occur at the right time . in man nearly all functions of the body including those influencing pharmacokinetic and pharmacodynamic parameters show marked daily variations . chronesthesyis the administration time of a drug based on the circadian or other bioperiodic rhythm , related to the rhythm in receptor number or conformation and rate - limiting steps in metabolic pathways . we can conclude that concerning the available data , there is clear evidence that the dose / concentration - response relationship of drugs can be significantly dependent on the time of day . thus , circadian time has to be taken into account as an important variable influencing a drug 's pharmacodynamics and/or its effects or side effects . authors contributed equally to this work and terken baydar is the corresponding author of this paper .

for many decades , researchers are aware of the importance of circadian rhythm in physiological / biochemical properties and drug metabolism . chronopharmacology is the study of how the effects of drugs vary with biological timing and endogenous periodicities . it has been attaching substantial attention in the last years . chronopharmacodynamics mainly deals with the biochemical and physiological effects of drugs on the body , the mechanisms of drug action , the relationship between drug concentration and effect in relation to circadian clock . in this review , we will focus on mammalian circadian pharmacodynamics and discuss new chronotherapy approaches . moreover , we will try to highlight the chronopharmacodynamics of cardiovascular drugs , anti - cancer drugs , analgesics and non - steroidal anti - inflammatory drugs ( nsaids ) and give some practical concerns for clinical pharmacists and pharmacy practitioners , concerning this issue .

INTRODUCTION Chronesthesy and chronopharmacodynamics Cardiovascular drugs Anticancer drugs Analgesics and non-steroidal anti-inflammatory drugs Some practical points for clinical pharmacists and pharmacy practitioners CONCLUSION AUTHORS CONTRIBUTION

PMC5140808

the vertebral column is a set of unstable joints , which is involved in a variety of movements , including extension , flexion , right - left bending , and trunk rotation1 . because of its unstable structure , prior research has them reported that about 80% of the global population has lasting complained of back pain or had a history of back pain , and about 20% of complained of chronic low back pain ( clbp ) over 6 months2 , 3 . since people aged ( 30 to 50 ) have a high prevalence of clbp , it is also becoming a socially and economically significant problem , and it occurs mostly in women aged 40 or older4 . lbp can be categorized into acute ( shorter than 3 months ) and chronic ( 3 months or longer ) by its duration . in lbp , two of the most frequent types of pain are spondylogenic pain , caused by the vertebral column and its appendicular structures , and neurogenic pain caused by problems in muscle - controlling nerves5 . these types of pain are caused by the paravertebral muscles and other types of muscles failing to provide vertebral stability , weakened vertebral bodies , disks , or ligaments between vertebral bodies , or imbalance between the lumbar extensor and flexor muscles6 . when imbalance between the abdominal muscles of the trunk and extensor muscles occurs , it triggers lbp and reduces the stability of the lumbar segments7 . in general , less than 5% of all cases actually require surgical therapy , and application of non - surgical therapy alone can reduce pain and restore the functions of the lumbar spine8 . in the 1980s , the main cause of lbp was believed to be weakness of the muscles supporting the vertebral column ; therefore , application of active rehabilitation exercise program to improve muscular and physical strength was considered the most ideal gold - standard for patients with clbp9 , 10 . however , since recent studies have reported that stability of the vertebral segments plays an important role in the mechanisms underlying lbp , rehabilitation exercises based on lumbar stability , or core stabilizer exercises , which can minimize instability of the vertebral segments and eventually decrease lbp , is actively being used to treat lbp11 , 12 . characteristically , such rehabilitation exercise not only strengthens the muscles around the spine and pelvis , including the abdomen , but also activates proprioception when the body makes dynamic movements . it is also considered a therapeutic element playing a crucial role in maximizing body coordination and lumbar stability through inter - neuromuscular facilitation13 . however , park and colleagues14 reported that the use of a simple lumbar stabilization exercise program , focusing on strengthening the isolated local muscles of the vertebral segments , as a rehabilitation exercise method for patients with clbp , would not ease lbp and restore vertebral functions . therefore , a strong correlation between muscular fitness factors and neuromuscular activation in reducing lbp and achieving functional restoration may be suggested . renkawitz et al.11 reported a strong correlation between restoration of muscular function in global muscles and deep trunk muscles and neuromuscular activation in lbp treatment . in other words , the complementation of two types of therapy may reduce pain , support mobility of the vertebral joints , and restore the muscular functions of the spine . in this respect , cailliet15 reported that rehabilitation exercise could play an important role in restoring tissues by actively extending tendons , ligaments , and joint membranes of joint tissues to facilitate blood circulation as well as improve muscular strength and flexibility . many studies have conducted exercises for improving each of the muscular functions , or lumbar stabilization exercise for patients with clbp16 , 17 . however , there has been little research on the application of both lumbar stabilization exercises and an active muscular fitness program in a mixed type of rehabilitation exercise . a study of the inter - group differences following the application of a complex rehabilitation program for both males and females would be significant for the development of a universal lbp therapy and an effective rehabilitation exercise program . this study aimed to determine the effects of a rehabilitation exercise program combining muscular fitness exercise and lumbar stabilization exercise on the lumbar isometric muscular strength ( ims ) of both male and female patients with clbp in their 30s , the age group participating most actively in socio - economic activities . twenty individuals , 10 males and 10 females , who visited the j oriental medical hospital in seoul participated in this study . the participants were patients with clbp who had had lbp for over 3 months and were receiving general oriental therapy . those with severe cardiovascular disease , those who could not walk , or those with symptoms of spinal tumor , spinal or disk infection , inflammatory disease , mental illness , or neurological disorder accompanied by motor disturbance were excluded . before conducting this experiment , only those who understood the purpose of this study , and indicated their active intention to participate in the experiment , after they had listened to explanation on the information and procedure of the experiment this study received approval from the institutional review board ( irb ) of dongshin university on april 27 , 2014 ( irb no : 201401-bm-006 - 02 ) . the participants physical characteristics are shown in table 1table 1.physical characteristics of the subjectsgroup age ( years)height ( cm)weight ( kg)bmi ( kg / m)female ( n=10)34.4 2.8163.2 2.753.0 4.120.1 1.8male ( n=10)35.3 2.9176.6 3.072.8 1.equipment used for the isometric muscular strength test shows the ims measurement system used to measure the lumbar extensor strength machine ( medx , ocala , fl , usa ) . to assess the lower back and extensor strength , the maximum isometric extensor strength was measured at lumbar joint flexion angles of 0 , 12 , 24 , 36 , 48 , 60 , and 72. all patients received a detailed explanation of the experimental procedure in advance , were checked for lbp , and were warned of possible accidents during the test or exercise . the participants thighs and lower limbs were fixed with knee , thigh , and pelvic restraints , and the foot board to prevent mobilization of leg strength during the lumbar extension strength measurement . beginning from the lumbar flexion of 72 , the participants were asked to extend their waist gradually upon the tester s order , with reference to pain and restricted joint angles , and to maintain the maximum muscular strength for 12 seconds when the maximum strength was exerted the upper resistance pad for 23 seconds , to measure extension . equipment used for the isometric muscular strength test the rehabilitation exercise program in this study was composed of warm - up , main exercise , and cool - down , and was conducted in three , 60minute sessions a week . the program had three stages , conditioning , improvement , and maintenance phases , and was a revised and complemented version of the complex rehabilitation exercise program developed by park and colleagues14 . table 2table 2.the exercise program performed by the patients with clbpphasecomplex rehabilitation program typerep / set ( week)considerationap ( week 14)ms leg flexion / extension dumbbell biceps curl dumbbell triceps extension calf raise lat pull down710 reps/2 sets non - weight bearing open kinetic chain exercise exercise in range without pain low back flexion and extension to minimize frequency and intensity of control , depending on fitness level continuous supervision . ice massage after exercisest quadruped arm / leg lifts with bracing gluteal squeezes pelvic tilt710 reps/2 sets:710 reps with57 s holdip ( week 58)ms medx exercise ( isotonic) crunch machine leg press supine squat ( non - weight bearing) multi - hip ( adduction , abduction , flexion , extension)710 reps/3 sets : medx 15 reps/1 set closed kinetic chain exercise eccentric exercise for strength enhancement return to adaptation phase , if pain occurred prohibit from overloading and over - repetition recommendation of minimal bed rest recommendation of continued independent ambulation avoid to high impact exercise such as running ice massage after exercisest hip rotation exercise with elastic band superman crunch on gym - ball side bridge on floor710 reps/2 sets : 710 reps with 510 s holdmp ( week 912)ms medx isotonic exercise standing squat ( weight bearing) push - up dumbbell lateral shoulder raise 1012 reps/3 sets : medx 15 reps/1 set weight - bearing exercise functional training , including agility light jogging continuous exerciserecommendatiost pnf d1-d2 side bridge on gym - ball reverse curl with gym - ball superman position on gym - ball prone pull ins on gym - ball710 reps/3 sets:710 reps with510 s holdclbp : chronic low back pain ; ap : adaptation phase ; ip : improvement phase ; mp : maintenance phase ; ms : muscular strength ; st : stabilization ; pnf d1-d2 : proprioceptive neuromuscular facilitation diagonal pattern 1-diagonal pattern 2 . * intensity=1 rm ( repetition maximum ) 5080% ( measurement of isotonic or dynamic strength ) ; type = circuit training ; rest=30 s / set shows the exercise type , frequency , intensity , time , and considerations of each phase . clbp : chronic low back pain ; ap : adaptation phase ; ip : improvement phase ; mp : maintenance phase ; ms : muscular strength ; st : stabilization ; pnf d1-d2 : proprioceptive neuromuscular facilitation diagonal pattern 1-diagonal pattern 2 . * intensity=1 rm ( repetition maximum ) 5080% ( measurement of isotonic or dynamic strength ) ; type = circuit training ; rest=30 s / set sas ver . the paired t - test was carried out to investigate gender differences before and after the intervention . analysis of covariance ( ancova ) was performed with prior values set as covariates to examine gender differences according to experimental treatment . when inter - group differences were found , a post - test was performed using the least squares method ( lsm ) . the significance level ( ) for testing the hypotheses of this study was 0.05 . the differences in ims before and after the intervention are shown in table 3table 3.changes in ims presented by gendervariablegrouppre - testpost - test%0female51.4 26.876.5 35.825.1 7.0**male87.3 18.8135.4 15.748.1 10.2**12female73.7 33.6106.2 51.232.5 8.6**male123.4 24.5179.0 25.055.6 12.9**24female95.1 38.6123.0 56.527.9 9.2*male151.9 35.9202.1 21.650.2 14.1**36female106.7 44.5133.5 56.626.8 6.9**male173.8 32.4219.1 25.145.3 13.5**48female117.1 48.8142.6 53.525.5 7.0**male190.6 32.6235.7 22.345.1 14.0 * 60female127.0 45.6154.4 55.827.4 7.7**male194.8 41.7246.4 25.751.6 13.8**70female137.6 52.6161.6 56.824.0 10.9*male209.6 30.2257.5 35.747.9 11.3**ims : isometric muscle strength ; % : percent difference between pre and post ; mean sd ; * p<0.05 , * * p<0.01 . there were increases in ims at every angle for both males and females , and there were statistically significant differences . ims : isometric muscle strength ; % : percent difference between pre and post ; mean sd ; * p<0.05 , * * p<0.01 the gender differences following experimental procedure as a main effect were statistically significant in ims at lumbar extensor strengths of 0 , 48 and 60. to detail the differences , lsm values are shown in tables 4table 4.lsm values of lumbar extension ims at 0grouplsmft - valuepost - hocfemale85.3 9.47.61**2.76*female < malemale126.6 9.4lsm : least squares method ; mean sd ; * p<0.05 , * * p<0.01 , 5table 5.lsm values of lumbar extension ims at 48grouplsmft - valuepost - hocfemale165.9 12.35.51**2.35*female < malemale212.4 12.3lsm : least squares method ; mean sd ; * p<0.05 , * * p<0.01 and 6table 6.lsm values of lumbar extension ims at 60grouplsmft - valuepost - hocfemale177.0 12.15.97*2.44*female < malemale223.8 12.1lsm : least squares method , mean sd ; * p<0.05 . lsm of ims at the angle of 0 , 48 and 60 by gender , as shown in tables 4 , 5 and 6 , and they demonstrate that ims was statistically significantly higher for males than for females . lsm : least squares method ; mean sd ; * p<0.05 , * * p<0.01 lsm : least squares method ; mean sd ; * p<0.05 , * * p<0.01 lsm : least squares method , mean sd ; * p<0.05 low back pain is a typical musculoskeletal disease caused by weakening or disharmony of the inner and outer muscles of the trunk and other tissues ( tendons , ligaments , joint membranes , etc . ) affecting spinal and pelvic mobility , and stabilization , mainly due to problems with the musculoskeletal structure . in particular , it varies from simple types caused by abnormal biomechanical problems to complex ones caused by a variety of factors , and has various symptoms , thus requiring very complicated therapies18 . as a safe method of treating lbp with relatively few side effects and reducing medical expenses , , it is believed that the development of a clinically usable rehabilitation exercise program for lbp therapy , is of itself significant . this study aimed to assess the effects of a rehabilitation exercise program composed of ims exercise and lumbar stabilization exercise on lumbar ims of the low back of 20 males and females in their 30s , diagnosed with clbp . muscular exercise for strengthening the trunk muscles is a very important intervention for patients with lbp . the normal functions of various types of muscles around the vertebral joints can provide stable support and enable smooth dynamic movement of the body19 . in a previous study , a lumbar segmental stabilization exercise elicited a statistically significant effect on the in strength of the muscles of the gluteus group20 . in another study , aquatic therapy exercise enhanced the low back muscle strength and reduced lbp of elderly females21 . this study assessed the lumbar ims at 0 , 12 , 24 , 36 , 48 , 60 , and 72 to determine the muscular fitness of patients with lbp and found there was a significant improvement across the entire range measured following the performance of the complex rehabilitation program . as for gender differences , males showed significantly greater improvement at 0 , 48 , and 60 than females . this gender difference can be considered as a difference in physiological reaction to resistance exercise . in other words , there is no gender difference in muscular strength before puberty ; however , in adulthood , females have lower muscular strength than males due to hormonal changes , weight , body composition , muscle mass , and transversal area of muscular fibers22 . jackson , shepherd , and kell23 reported that resistance exercise performed for 16 weeks reinforced the lumbar muscular strength of middle - aged people with clbp . like this study , other prior studies have reported that resistance exercise beginning with low intensity and with a gradually increasing intensity and number of sessions is effective at improving the muscular strength and lowering the rate of repeated injury among in patients with clbp24 . it is considered that muscular function improvement and lumbar stability exercise play important roles in controlling spinal segments and providing stability in the alleviation of lbp11 , 12 . any prior studies have reported that effective rehabilitation exercise activates functions of the intersegmental muscles , such as the multifidus , rotators , and abdominis muscles , which contribute to the functioning of the spine , improving stability and balance control of the body25 . thus , it can be emphasized that scientific verication of an exercise program is crucial for lbp therapy and recurrence . as for prior research concerning specific exercise programs , park and colleagues14 indicated that an exercise program that simultaneously strengthens the deep abdominal muscles and muscles of the trunk is the most ideal method for maintaining spinal stability and physical balance , and the american college of sports medicine ( acsm)26 recommends that it is necessary to perform lumbar and abdominal muscular strength exercises more than once a week , and flexibility exercise for improving the mobility range of vertebral joints for more than three sessions , 10 seconds per session . it is also recommended that priority should be given to simultaneous maintenance of the lower limbs rather than simple reinforcement of muscular strength , and that spinal correction exercise for improving postural balance should be performed in addition to improving lumbar muscular functions . in conclusion , this study conducted a complex rehabilitation exercise for 20 patients ( 10 females , 10 males ) with clbp who were in their 30s in 60-minute sessions , 3 times a week for 12 weeks , to determine the changes elicited in lumbar ims by gender . males showed greater improvement than females at the angles of 0 , 48 , and 60. therefore , a 12-week complex rehabilitation program for muscle strengthening aimed at patients with clbp appears to be effective at improving lumbar muscular strength .

[ purpose ] the purpose of this study was to assess the effects of complex rehabilitation training on chronic low back pain . [ subjects and methods ] complex rehabilitation training for lumbar isometric muscle strength was conducted for 12 weeks for males ( n=10 ) and females ( n=10 ) with chronic low back pain , who were aged in their 30s . [ results ] isometric strength of lumbar extension was increased post - test , and significant differences were found between males and females , at medx angle of 0 , 48 , and 60 . [ conclusion ] this study showed that complex rehabilitation training had a beneficial effect on the muscle strength of lumbar extension in patients with chronic low back pain .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4175228

children with cerebral palsy ( cp ) have a neurodevelopmental disorder due to a nonprogressive lesion of the immature brain occurring early in infancy or during the fetal term1 , 2 . motor disorders of children with cp are related to primary deficits ( such as spasticity , muscle weakness , reduced coordination , and a loss of selective motor control ) and secondary deficits ( such as muscle contracture and bone deformities)3 , 4 . these changes can affect voluntary muscle recruitment and lead to impairment of motor ability . compared with children with normal development ( nd ) , children with cp have various muscle recruitment patterns and magnitudes5 . abnormal gait is a common problem in children with cp . because of motor weakness and poor voluntary motor control , crouched gait or diplegic gait is an important functional biomarker in children with spastic diplegic cp6 , 7 . in addition , gait in children with cp is characterized by a slower walking speed , a shorter - stride length , and more time spent in double support8 . due to the abovementioned problems , a general decrease in physical activity as well as walking capacity has been observed in children with cp . van den berg - emons et al.9 and bjornson et al.10 reported that school - age children with diplegic cp were less physically active than nd children . these characteristic features influence quality of life of children with cp by reduction of daily life activities and independent mobility11 . although it is well recognized that children with cp have poor gait capacity and physical activities , the majority of the scientific literature in children with cp has focused on kinetics and kinematics in assessment of gait analysis . in addition , previous studies have not included a direct comparison of school - age children with cp and age - matched nd children . gait capacity limitations in children with cp appear to be related to the ability to participate in day school activities , such as playground games and moving to other areas of the school12 . increasing the knowledge concerning the difference in gait capacity between children with spastic diplegic cp and age - matched nd children can be helpful for persons involved in the care of these children . therefore , the purpose of this study was to determine the differences in spatiotemporal gait parameters between children with spastic diplegic cp and nd children . eight children with spastic diplegic cp and eight nd children participated in this study . children with cp had a gross motor function classification ( gmfc ) system level between i and ii and removed their prescribed ankle - foot orthosis during the experiment . the following inclusion - exclusion criteria were used for selection of children with cp : ( 1 ) children with cp had to have a clinical diagnosis of spastic diplegic cp ; ( 2 ) children with cp had to be able to walk without use of assistive devices ( i.e. , walker , cane , and ankle - foot orthosis ) ; and ( 3 ) children with cp had to have not undergone orthopedic surgery within at least one year and six months after botulinum toxin type a ( botox ) injections prior to the experiment . all parents consented , and the children agreed to participation in the experiment ; they provided written informed consent prior to their participation in the study in accordance with the ethical standards of the declaration of helsinki . objective gait analysis was performed using the orthotrak 6.4 system ( motion analysis , santa rosa , ca , usa ) , and marker trajectories were obtained using an evart 6.1 real - time tracking system ( motion analysis , santa rosa , ca , usa ) with an eight - camera system ( hawk system , motion analysis , santa rosa , ca , usa ) . data from two force platforms embedded into a 10 m walkway were collected . for gait analysis , passive markers were attached with adhesive tape to the following locations of both lower limbs : the anterior superior iliac spine , sacrum , anterior aspect of the mid thigh , lateral femoral epicondyle of the knee and mid calf , lateral malleolus , base of the heel , and dorsum of the foot between the second and third metatarsals . the spatiotemporal parameters included walking velocity , cadence , stride length , and percentages of single- and double - limb support . the gmfm-66 ( gross motor functional classification 66 ) is a standardized observational measurement designed for assessment of changes in five important gross motor function domains over time in children with cp . the five gross motor function domains , which include 66 testing items , include lying and rolling ( four items ) ; sitting ( 15 items ) ; crawling and kneeling ( 10 items ) ; standing ( 13 items ) ; and walking , running , and jumping ( 24 items ) . the scoring system ranges from 0 ( does not initiate ) to 3 ( complete)13 . high levels of validity , reliability , and responsiveness have been demonstrated in evaluations of motor function using the gmfc in children with cp14 . an independent t - test was used for determination of differences in variables in terms of the baseline data ( i.e. , age , height , weight , and foot length ) and spatiotemporal parameters ( i.e. , gait velocity , cadence , stride length , step width , stance phase , swing phase , and double - limb phase ) between children with cp and nd children . the baseline demographic characteristics of the subjects enrolled in the study were as follows . the mean age , height , and weight of the children with cp were 8.20.75 years , 125.67.05 cm , and 30.53.35 kg , respectively , and those of the nd children were 9.10.44 years , 137.12.78 cm , and 34.62.79 kg , respectively . four of the children with cp were classified as gmfc level i , four children were classified as gmfc level ii . data ( meansd ) for the spatiotemporal gait parameters of both the children with cp and the nd children are shown in table 1table 1.comparison of spatiotemporal gait parameters in the cp and nd groupsparameterscp children ( n=8)nd children ( n=8)walking velocity ( m / min)63.99 11.22 * 107.38 5.14cadence ( step / min)89.39 8.04 * 115.44 3.17stride length ( cm)81.25 12.42 * 111.31 3.20step width ( cm)11.80 1.72 * 7.38 0.72rt . tdls ( % /unit)18.19 3.06 * 8.88 0.67lt . tdls ( % /unit)15.33 2.73 * 8.59 0.55 * significant difference between the cp and the nd children ( * p<0.05 ) . cp , children with cerebral palsy ; nd , normal developmental child ; sls , single - limb support ; idls , initial double - limb support ; tdls , terminal double - limb support . significant differences in terms of spatiotemporal gait parameters were observed between the two groups . the scores for walking velocity , cadence , and stride length in the children with cp were lower compared with the nd children , whereas the score for step width was higher in the cp children compared with the nd children . the periods of right and left single - limb stance in the children with cp group were shorter compared with the nd children , whereas those for right and left double - limb stance were longer in children with cp compared with the nd children . * cp , children with cerebral palsy ; nd , normal developmental child ; sls , single - limb support ; idls , initial double - limb support ; tdls , terminal double - limb support the aim of this study was to describe differences in spatiotemporal gait parameters between children with spastic diplegic cp , who had gmfc system levels of i or ii , and nd children , and to determine the extent of the differences . our findings showed significantly lower spatiotemporal gait parameters , including walking velocity , cadence , and stride length in children with spastic diplegic cp than in nd children . step width was larger in children with cp due to poor balance and gait instability . in addition , significant differences in proportions of the stance phase ( single- and double - limb stance ) were observed in children with spastic diplegic cp compared to nd children . the duration of single - limb support in children with spastic diplegic cp was shorter , and that of double - limb support was longer . our findings are consistent with those of previous studies reporting deteriorated gait function and altered gait pattern in children with spastic diplegic cp8 , 15 . the findings of the current study showed that walking velocity , cadence , stride length , and step width of children with cp with a gmfc of between i and ii were a level of 60% , 77% , 73% , and 160% , respectively , of those of nd children . because of motor weakness and poor voluntary motor control , children with cp use a wider step width than nd children16 , suggesting that children with cp may choose a wider base of support in order to stabilize the center of mass . in addition , step width showed correlation with walking velocity , cadence , and stride length . thus , children with a wider step width tend to have greater difficulty in gait performance . the percentages of right and left double - limb support and of right and left single - limb support during stance phase were 188% and 179% higher and 83% and 82% lower , respectively , compared with the nd children . this is because reduced hip adduction in the stance phase increases the internal abduction moment of the support limb . previous studies have reported that children with spastic diplegic cp show weakness of the hip abductor muscles17 , 18 . thus , the gait pattern would be unstable , and less time would be spent in single - limb support . children with spastic diplegic cp with a gmfc level of i are expected to walk independently indoors and outdoors and to go up and down stairs . children with a gmfc level of ii hold onto a rail when walking on stairs and are limited with respect to walking long distances . thus , the differences between the school - age 7 to 12 years old children with spastic diplegic cp ( gmfm levels i and ii ) and age - matched nd children included limitations in walking outdoors and inside the school , walking on uneven surfaces , and walking in crowded places . our results provide objective evidence of distinct differences in spatiotemporal gait parameters between children with spastic diplegic cp with a gmfc level of i or ii and nd children and would be helpful to persons involved in the care of these children . the therapeutic approach to management of children with cp , who have a wide base of support and decreased capacity to shift weight , should be considered for improvement of ability to maintain single - limb support and for improvement of ability to shift weight in order to reduce the period of double - limb support . due to the small number of children with cp in the present study , some limitations could be raised and should be taken into account when interpreting the data . in addition , because our study was only conducted in school - age children with spastic diplegic cp with a gmfc system level of between i and ii , the results may not be generalized to those with more severe forms of cp . however , our study has meaningful significance in that it suggests quantitative value for spatiotemporal gait parameters for school - age children with spastic diplegic cp compared with nd children . therefore , conduct of future studies may be needed in order to clarify this issue .

[ purpose ] the purpose of this study was to determine the differences in spatiotemporal gait parameters between children with spastic diplegic cp and children with normal development ( nd ) . [ subjects and methods ] sixteen children ( eight children with spastic diplegic cp and eight nd children ) were recruited for participation as volunteers in this study . the children with cp had a gross motor function classification ( gmfc ) system level of between i and ii . [ results ] walking velocity , cadence , stride length , and step width of children with cp with a gmfc of between i and ii were a level of 60% , 77% , 73% , and 160% , respectively , of those of nd children . the percentages of right and left double - limb support were 188% and 179% higher , respectively , and the proportion of single limb support was shorter by 83% and 82% . [ conclusion ] our results provide objective evidence of distinct differences in spatiotemporal gait parameters between children with spastic diplegic cp with a gmfc level i or ii and nd children and would be helpful to persons involved in the care of these children .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC2426772

the isolation of pure populations of cells from heterogeneous cell suspensions is an essential part of clinical as well as basic research . the diagnostic test for hiv infection , for example , relies on the separation of human lymphocytes from whole blood . isolation of pure cell populations is an essential part of research efforts to understand fundamental aspects of the body s response to injury ( feezor et al 2004 ; cobb et al 2005 ) . more recently , with major advances in stem cell biology , the isolation of rare cells has become an active area of research . for example , islet 1 + cells that appear to be resident cardiac progenitor cells have been identified in mouse and human hearts ( laugwitz et al 2005 ) . these cells are present in very small numbers , ~100 islet 1 + cells among 10 heart cells . the progenitors are currently isolated by pre - plating of the heart cell suspension for 1 hour , after which fibroblasts and some islet 1 + cells remain attached to the tissue culture plastic . after 46 days in culture , the progenitors make up only 0.5% of the total cell population found in the plates . development of robust and efficient methods for isolating rare cells such as these would clearly represent a major contribution to stem cell biology . miniaturized cell separation devices offer many advantages over conventional separation techniques ( eg , density gradient centrifugation ) , such as small sample volumes , portability , low cost , improved sterility , and potential for integration with analytical techniques . many of these technologies are derived from the semiconductor industry or from advances in synthetic chemistry . cell separation techniques can be broadly classified into two categories : techniques based on size and density , and techniques based on affinity ( chemical , electrical , or magnetic ) . in this review , rather than providing a comprehensive review of all cell separation techniques , we provide representative examples from each category with emphasis on micro- and nanotechnology . since cells are of the order of microns in size and usually handled in suspensions , nearly all of the methods described here are microfluidic . however , some of these techniques , in particular the magnetic separation techniques , also incorporate nanoscale elements . purity and throughput are important metrics for any separation process , and it is often impossible to attain high levels of both . several novel size - based separation processes are being employed in micro- and nanoscale devices . these devices are compact , simple , and usually do not require additional energy sources or additional equipment . most of the devices force a fluid containing a heterogeneous particle population through a series of channels or obstacles of varied size . unlike macroscale size - based separation approaches such as the use of cell strainers , the microscale geometry of the flow channels in these devices ensures that fluid flow is laminar . the main advantage of the size - based approach is that it does not require the presence of cell - specific markers or proteins to achieve separation . hence this approach can be used to isolate stem cells and other rare cells that do not express known markers . furthermore , since the devices do not contain any biological markers or proteins , they have long shelf - life and are easily transportable . however , one of the major concerns with this approach is maintaining cell viability as the cells pass through the microfluidic device . huang et al ( 2004 ) describe a technique that allows for separation of micron - sized particles with a resolution of 0.1 m . in this approach , individual fluid streams can be made to flow along confined lanes in a deterministic manner simply by allowing them to pass through an array of obstacles . the assumption is that a fluid stream will bifurcate when it encounters an obstacle , and thereby sort the particles it brings with it ( figure 1a ) . as indicated by the arrows in the figure , the fluid streams travel around the obstacles and become confined to one of three lanes . depending on the size of the particles in the stream and the obstacle parameters , the stream can enter one of two modes of displacement . if the particles are smaller than the width of the lane they are flowing in , the particles zigzag back and forth around the obstacles ( zigzag mode ) , whereas if they are larger than the lane width , they collide with the obstacles and keep displacing in one direction ( displacement mode ) . by manipulating the obstacle parameters , such as their spacing and diameter , as well as the amount by which they are staggered in each subsequent row of the patterned array , micron - sized particles can be sorted rapidly ( in a few minutes ) with high resolution and with an uncertainty less than 1% of the particle size , or , of the order of tens of nanometers . such resolution is similar to that of other sorting techniques such as hydrodynamic chromatography and quasi - elastic laser light scattering , and considerably better than that of techniques such as cell straining and density gradient centrifugation . as a proof of concept , the authors used the technique to sort fluorescent polystyrene spheres , as well as circular plasmid dna . some limitations of the technique include the requirement of laminar flow and the possibility of random diffusion between adjacent lanes . nanofabrication may allow for improved resolution and sorting of supramacromolecules such as viral particles and protein complexes . ( a ) a stream of fluid flowing at low re ( ~10 ) is forced perpendicular to a series of obstacles of defined size and spacing ( see arrows ) . if particles are smaller than the lane width , they continually zigzag between the obstacles , returning to their original lane assignment after traversing several rows of obstacles ( zigzag mode ) . however , when the particles are larger than the lane width , they collide with the obstacles and displace only in one direction ( displacement mode not shown ) , allowing for separation to occur . ( b ) a series of channels of successively smaller width is microfabricated thus creating a cell sieve . mohamed et al ( 2004 ) have designed a device that uses a relatively simple size - based separation approach to isolate rare cells , such as metastatic cancer cells , from peripheral blood . the device contains four successively narrow arrays of parallel channels ranging from 20 m down to 2.5 m in thickness ( figure 1b ) . the device essentially achieves separation by acting like a sieve , trapping larger cells such as mononuclear cells and neuroblastoma cells further upstream in the larger channels , and allowing smaller cells such as erythrocytes to pass through . the authors experimented with several designs of varied channel width and height before identifying the critical dimensions required to trap the cells of interest . the channel width and height were tuned to ensure that the larger , rare cells ( in this case , neuroblastoma cells or mononuclear cells ) were always trapped while allowing the smaller , bulk cells ( in this case , erythrocytes ) to always pass through . thus , the approach can be applied to separation systems of mixed cell populations provided that the two cell types being separated are sufficiently different in size . to make the process more cost effective , a combination of soft lithography ( pdms , poly(dimethylsiloxane ) ) and polymers ( polyurethanes ) were used for fabrication . when cultured neuroblastoma cells were mixed with either plain medium or peripheral blood and passed through the device , the larger neuroblastoma cells were consistently retained in the channels 10 m wide and 20 m deep while erythrocytes traversed through the device completely . similarly , mononuclear cells in a sample of whole blood were trapped in channels 2.5 m wide and 5 m deep . while the technique is not without its limitations , including the issue of nonspecific cell adhesion to the channel walls , the technique is more effective in terms of cost and time than conventional cell sorting techniques such as fluorescence - activated cell sorting ( facs ) or magnetic - activated cell sorting ( macs ) . besides using a series of obstacles to achieve size - based cell separation , cells shevkoplyas et al ( 2005 ) have developed a biomimetic microfluidic device to facilitate separation of leukocytes from erythrocytes in whole blood samples . the device does not require a power source ( except for a small pressure gradient created by a water column to facilitate blood flow ) , and achieves efficient autoseparation in a single pass using low sample volumes ( ~l ) . it is biomimetic in that it contains a series of bifurcating , rectangular microchannels , emulating the natural structure of blood vessels in vivo . furthermore , it relies on the natural phenomenon of plasma skimming within a 2d channel to enrich the leukocyte population . when the erythrocytes and leukocytes interact and collide , the erythrocytes , which are smaller and more deformable than leukocytes , preferentially migrate toward the center of the channel , forcing the leukocytes to migrate to the outer wall of the microchannel . by diverting the leukocyte - rich plasma layer through several bifurcations , the authors achieved a 34-fold amplification in the leukocyte - to - erythrocyte ratio compared with the inlet concentration . the device can prove particularly useful in applications where high concentrations of leukocytes or their dna and rna are required for analysis . differential motility in laminar flow was used by cho et al ( 2003 ) to separate motile sperm cells from non - motile sperm cells . central to their design is the assumption that viable sperm cells are motile and will therefore migrate against a laminar fluid stream . in contrast , non - motile sperm simply follow the fluid stream lines along with other debris or round cells in the sample . the motile sperm will move across the fluid stream and exit via a different outlet at the bottom of the device , distributing themselves along the width of this outlet , while the non - motile sperm will exit at the outlet situated at the top of the device . the design does not require a power source since it utilizes a height difference between the inlet and outlet as well as surface tension of the fluid stream to create a gravity - driven pump , facilitating a constant fluid flow rate , regardless of the fluid reservoir volume . the authors demonstrated that motile sperm can be purified to levels of nearly 100% . additionally , the yields of motile sperm at the outlet compared with the inlet were significantly improved and comparable with other conventional separation techniques such as direct swim - up , swim - up from centrifuged pellet , and density gradient separation . cho et al have proposed several useful applications for the technique , such as home - based screening test for infertility , vasectomy , vasectomy reversal , and sperm toxicology tests . further improvements to the geometry and configuration of the chip , such as serial connections , may lead to even higher yields . these systems typically create a hydrodynamically focused stream of cell suspension which is interrogated using a laser beam and an array of photodetectors . fluorescent labels are attached to one or more cell types in a heterogeneous suspension and the cells are sorted individually based on ( a ) how they scatter the incident laser light and ( b ) the wavelength of light that they emit . a recent review by huh et al ( 2005 ) describes a number of microfluidic systems based on this principle . an important advantage of microfluidic facs systems is their low cost compared with conventional systems . in the context of medicine and point - of - care diagnostics furthermore , microscale facs systems have been shown to operate comparably to their macroscale analogs in terms of purity and throughput ( wolff et al 2003 ) . fu et al ( 1999 ) have described a device made of silicone elastomer and glass with embedded platinum electrodes to direct the cells by electro - osmotic flow . an external laser and photomultiplier tube ( pmt ) interfaced with a computer are used to excite and detect fluorescence . flow of the cell suspension is controlled by changing the electro - osmotic potentials in response to the pmt signal . the forward algorithm , cells that normally flow from the inlet to the waste reservoir are redirected to a collection channel if the fluorescence is above a preset threshold . with the reverse algorithm , cells flow at a high rate from the inlet to waste until the detection of fluorescence from a target cell induces the flow to reverse direction to allow a second detection step , followed by flow to the collection channel . forward algorithm is analogous to the mode of operation of most conventional facs systems , whereas the reverse is a novel algorithm that can not be implemented in conventional systems . a key advantage of the reverse algorithm is that it can be used to make more than one measurement on individual cells . this device was used to separate a mixed population of fluorescent and non - fluorescent escherichia coli cells . in a subsequent publication , the same group ( fu et al 2002 ) described a more advanced version of this device which consists of integrated microvalves and micropumps . the incorporation of these components on - chip allows for the elimination of some macroscopic components and better control of functions such as sample dispensing and recovery , and flushing . with the same sample of mixed cell populations ( fluorescent and non - fluorescent mixture of e. coli cells ) , this device allowed enrichments of up to 90-fold with a throughput of 10 cells / hour . a further step towards a fully integrated microfluidic facs system has been taken by krger et al ( 2002 ) . in their device , the laser and detectors are incorporated along with the microfluidic components in a single chip . the only macroscopic parts of this system are a laptop computer , charge - coupled device ( ccd ) camera , and syringe pumps . another example of an advanced microfluidic facs system with integrated waveguides and a cell culture chamber has been described by wolff et al ( 2003 ) . with this device , the authors were able to separate fluorescent latex beads from chicken red blood cells at high throughput ( 12 000 cells / second ) . this high rate was made possible by a high - speed hydrodynamic valve with a response time of 2.5 milliseconds , which was interfaced with the pmt . the authors also developed a second - generation device ( figure 2 ) designed for improved hydrodynamic focusing and on - chip cell culture . this device was used to separate normal yeast cells from yeast cells containing green fluorescent protein . the detected cells were directed to the culture chamber by the high - speed valve and allowed to grow and divide for several days with continuous flow of fresh medium . the incorporation of the culture chamber illustrates how microfluidic facs systems can reduce the risk of sample loss due to external handling or dead volumes , a feature that would be particularly relevant for the isolation of rare cells . cells flow through a channel into an observation region . upon detection of a target cell , a high - speed hydrodynamic valve switches fluid flow to send the cell into the holding / culturing chamber . source : wolff a , perch - nielsen ir , larsen ud , et al . 2003 . integrating advanced functionality in a microfabricated high - throughput fluorescent - activated cell sorter . abbreviations : pmt , photomultiplier tube ; facs , fluorescence - activated cell sorting . the imag cell separation system ( bd biosciences , san jose , ca , usa ) is one example of several available separation systems . when exposed to a mixed cell population , the magnetic beads attach to the surface of desired cells via antibody antigen interaction . the desired cell subpopulation can then be separated in the presence of a strong magnetic field either in separation columns or on integrated microchips . magnetic separation is applicable only to cells that can be separated by specific surface antigens . for cells that are commonly distinguished by intercellular proteins ( eg magnetic sorting is of particular interest since it can easily be miniaturized and utilized in applications where small sample volume is required . this is the main advantage of microscale magnetic separation over the well established and efficient macroscale analogs . the major challenge in the design of microscale magnetic separation systems , which is the focus of most studies , is achieving efficient separation in a continuous ( flow through ) system . while several examples of microscale magnetic separation systems have been described in recent years , macroscale systems remain more attractive in terms of purity ( > 95% ) and throughput ( ~ 10 cells / hour ) ( thiel et al 1998 ) . deng et al ( 2002 ) have developed a magnetic micro - filtration system for separation of magnetic beads from non - magnetic beads with up to 95% efficiency . in this set - up , nickel posts 10 m in diameter were positioned in a microfluidic channel . a heterogeneous suspension of beads on the order of 1 m was flowed into the channel in the presence of an external magnetic field that magnetized the nickel posts . as a result , the magnetic beads adhered to the posts while nonmagnetic beads were removed by the fluid flow . upon removal of the external magnet and while keeping flow velocity unchanged , the magnetic beads were removed from the microfluidic channel . a heterogeneous particle population can be separated using a combination of flow and magnetic field in the process called magnetophoresis depending on the particle size and magnetic properties , the particles were deflected into the magnetic field and carried into different outlet channels . although the two systems described were not tested with cells , they appear very promising for cell separation applications . the combination of magnetic microbeads and external magnetic field was utilized in a y - shaped microfluidic device by furdui and harrison ( 2004 ) to separate immunogenic t cells from whole blood with an efficiency of up to 40% ( figure 3a ) . in the first step , paramagnetic beads coated with protein - a / anti - human cd3 were flowed into the micro - device and immobilized using an external magnet . subsequently , whole blood was flowed over the bed of magnetic beads , resulting in the capture of t cells . upon removal of the external magnet , the t cells were flushed out of the microfluidic device and utilized for pcr analysis . while this purity appears low in comparison with standard methods , such as separation using antibody - coated erythrocytes in conjunction with ficoll - hypaque density gradient centrifugation ( > 90% efficiency ; wilson et al 1976 ) , the microfluidic approach has the advantage of being easy to integrate with downstream analysis systems . magnetic cell separation and sorting . ( a ) y - shaped device used to separate immunogenic t cells from whole blood in three steps . paramagnetic protein a / anti - cd3-coated paramagnetic beads are flowed into the microdevice and immobilized using an external magnet . subsequently , whole blood is introduced over the bed of magnetic beads , resulting in the capture of t cells . upon removal of the external magnet , ( b ) combination of fluid flow ( arrows ) and ferromagnetic strips used to separate leukocytes on planar surfaces . bright dots : time - lapse image of a tagged leukocyte moving along a ferromagnetic strip ( left ) . untagged red blood cells are moving along the direction of the fluid flow ( right ) . the matrix consists of two wire meshes superimposed at 180. source : lee h , purdon am , westervelt rm . magnetic cell separation can also be achieved on planar surfaces , as demonstrated by inglis et al ( 2004 ) . leukocytes were separated from red blood cells by flowing the blood over the surface coated with ferromagnetic strips . the cells were labeled with anti - cd45 conjugated superparamagnetic nanoparticles ( 20100 nm diameter ) such that each positive cell carried around 5000 particles . the strips were positioned at an angle of 9.6 relative to the direction of fluid flow , causing the deflection of labeled cells ( figure 3b ) . magnetic labeling can also potentially enable precise manipulation and positioning of cells at the desired location . to prove this principle , lee et al ( 2004 ) constructed a microelectromagnetic matrix by positioning two wire meshes on top of one another in a cross - pattern ( figure 3c ) . the electric current in each wire was independently controlled via a computer , enabling precise control of the magnetic field at each point in the grid . with this set - up , the authors were able to separate nonviable yeast cells from viable cells by moving the latter individually to a distant location within the grid in a controlled fashion . the solution containing the live / dead - stained , magnetic bead - bound yeast cells was introduced into the microfluidic channel , integrated with the wire mesh . one viable yeast cell and two nonviable cells were initially trapped by a single magnetic field peak . subsequently , the single peak was split into two smaller peaks ; one of the peaks held the nonviable cells and the other one held the viable cell . the magnetic peak that held the viable cell was then moved by adjusting the current in the wires , so that the viable cell was moved away from the nonviable cells . adhesion - based cell separation systems are akin to chromatography columns where a mixture is passed through a column packed with beads or other materials capable of binding to the undesired constituents of the feed . in cell separation , antibodies immobilized on surfaces are used for binding . an important advantage of this technique is that it can be used to separate cell populations that have the same size and/or density , such as subpopulations of human lymphocytes . another advantage of this approach is that there is no need for preprocessing incubation of the starting cell mixture with fluorescent or magnetic antibody tags . conventional macroscale adhesion based systems are described by the term cell affinity chromatography ( cac ) . these systems can generally provide high purity ( > 95% ) and high throughput ( 1010 cells / hour ) ( mandrusov et al 1995 ; putnam et al 2003 ) . a major limitation of these systems is their packed bed design , which maximizes the surface area to volume ratio but also results in long residence times ( in the order of 12 hours ) ( mandrusov et al 1995 ; putnam et al 2003 ; ujam et al 2003 ) . microfluidic cac systems provide high surface areas per unit volume but their small overall volume keeps residence times short ( order of minutes or less ) ( murthy et al 2004 ; sin et al 2005 ) . microfluidic cac systems designed to separate t and b lymphocytes from mixed suspensions have been described by murthy et al ( 2004 ) and sin et al ( 2005 ) . these investigations employed microfluidic chambers made of glass and pdms , whose interior surfaces were functionalized with antibodies capable of binding to surface antigens on the surface of the target cell type . both investigations used t and b lymphocyte cells lines , molt-3 and raji , to create the mixed suspensions , and anti - cd5 and anti - cd19 antibodies to capture the respective cell types . in both reports , the cells captured on the surface were extremely pure ( > 97% purity ) . nonspecific binding of the undesired cell type was overcome by attaching poly(ethylene glycol ) ( peg ) on the surface along with the antibody molecules ( murthy et al 2004 ) . in these approaches , while the number of cells that can be captured was limited by the relatively small surface area of each device , the authors proposed that throughput can be increased by designing a device with larger surface area or by running several devices in parallel . in the above two reports , the antibodies used for separation were specific to the desired cell type . anti - cd5 binds to the t cell line but not the b cell line ; and anti - cd19 binds to the b cell line but not to the t cell line . but what about situations where there is no unique cell surface receptor that distinguishes the target cell type from other cells in the mixed suspension ? chang et al ( 2005 ) have described an adhesion - based microfluidic separation technique that offers a way to address this challenge . this technique emulates the physiological process by which leukocytes from the blood stream are captured by blood vessel walls during an inflammatory response . blood vessel walls achieve this capture by presenting a variety of adhesion proteins ( such as selectins ) to the rapidly passing blood cells and selectively capturing only the cells that bear complementary ligands . the authors designed microfluidic devices out of silicon and glass where the capture surfaces were square and rectangular posts arranged in square and offset arrays , respectively . in this respect , their device is similar in design to that of huang et al ( 2004 ) and offers an interesting comparison even though the mode of separation is different ( adhesion - based in chang et al and size - based in huang et al ) . chang et al coated the post surfaces with e - selectin igg molecules and examined cell capture using hl-60 and u-937 myeloid cell lines , both of which express ligands for e - selectin . with the help of flow experiments as well as finite volume simulations , the authors observed that the movement of cells through their device occurs in three phases : ( 1 ) rolling , ( 2 ) transient capture on the trailing surface of the posts , followed by ( 3 ) detachment and subsequent flow . the authors postulate that each of these three phases is influenced by the strength of bonds between the cells and the selectin molecules on the posts . in the square array device , they then detached and always moved to the very next post downstream . in the offset array devices , however , the cells traveled arbitrary distances following the capture and release events , not always moving to the very next array . although this report does not include experiments with mixed cell suspensions containing both cell types , the authors did measure a significant difference in transit time between the two cell types in their square array device : 1.4 0.8 mm / second for hl-60 and 2.7 1.4 mm / second for u-937 . this difference could conceivably be utilized to separate cell populations that are indistinguishable in size and surface receptor expression . adhesion - based separation can also be performed in a static microfabricated system , as opposed to a fluidic system . revzin et al ( 2005 ) have described the fabrication of microwells made of peg for the isolation of lymphocyte subpopulations . these wells are functionalized with antibodies and are capable of capturing target cells bearing complementary antigens with high selectivity . cd5 + t lymphocytes ( molt-3 cell line ) adhered preferentially to microwells coated with anti - cd5 ( 95% of the available wells were occupied ) and showed only minimal adhesion to microwells coated with avidin and anti - cd19 ( well occupancies were 5% and 13% , respectively ) . with a microwell size of 15 m 15 m , the authors observed that each well contained a single lymphocyte ( figure 4a ) . the significance of this capability was demonstrated by the selective removal of individual t lymphocytes from an array of microwells by laser capture microdissection ( lcm , figure 4b ) . lcm is technique whereby individual cells from different array locations are made to adhere to an adhesive film placed on top of the substrate surface by a pulsed and focused laser beam . the adhesive film is mounted on a cap that is subsequently removed from the substrate surface . the microwell platform offers a simple and effective method of isolating cell subpopulations based on antigen expression . when combined with lcm extraction , this technology could potentially be employed to extract useful proteomic or genomic data from the selected cells . ( a ) poly(ethylene glycol ) ( peg ) microwells containing individual t and b lymphocytes . this type of capture allows subsequent extraction of individual cells by laser capture microdissection ( shown schematically in ( b ) ) . source : revzin a , sekine k , sin a , et al . 2005 . most electrophoretic separation processes employ a process known as dielectrophoresis ( dep ) , whereby particles with an induced electric polarization , such as dipoles , quadrupoles , and octopoles , can be trapped within a nonuniform electric field . negative dep forces cause polarized objects to be repelled away from an electrode , while positive dep forces attract objects toward an electrode . since dep depends on the existence of a gradient in an electric field , an alternating current ( ac ) voltage is usually employed . the response of a cell to dep - mediated forces is influenced by cell type , its density , and even its metabolic and physiologic state , making these important parameters for separation . there are essentially three variations of dep currently being employed for micro- and nanoscale devices : dep retention , dep migration , and dep - field flow fractionation ( dep - fff ) . dep retention ( or dep affinity ) is used to hold cells against a fluid flow , so that cells only weakly influenced by dep forces are carried along with the fluid , while cells held more strongly by dep forces can be eluted later . dep migration is similar to dep retention , but instead it utilizes dep to cause cells to migrate to different regions of an electrode . finally , dep - fff uses dep forces to differentially levitate cells against gravity in a fluid flow profile , such as a parabolic flow profile . this causes cells with different dielectric properties to travel at different velocities along the channel . like magnetic separation , the primary advantage of microscale electrophoretic separation systems is the ability to handle small sample volumes . voldman et al ( 2002 ) have developed a microfabricated separation system capable of trapping and sorting single cells on the basis of their response to certain stimuli . the authors describe it as a microfabrication - based dynamic array cytometer ( dac ) , since it allows cells to be physically arrayed into dep - based traps , and dynamically probed and sorted thereafter . an array of dep traps , each comprising four trapezoidally arranged electrodes of opposite polarity , was fabricated to create a non - uniform , quadrupole electric field ( figure 5a ) . cells became electrically polarized in the presence of the quadrupole field and were physically trapped within a potential energy well against a fluid flow ( figure 5b ) . a high frequency ( ~mhz ) ac field was chosen to minimize power dissipation and heat - induced damage to the cells , to prevent transmembrane polarization of cells , and also to minimize corrosion of the electrodes . the cells ( hl-60 cell line ) were fed into the chip via a reservoir , and then exposed to a stimulus ( calcein ) . they were then individually loaded into the traps and optically interrogated over time for their response to the stimulus ( calcein loading ) by optical microscopy . each of the traps was electrically addressable , allowing for manipulation and release of a single cell or multiple cells simultaneously . thus , based on their responses , the cells could be sorted by turning on or off each individual trap . the authors demonstrated this capability in calcein - labeled hl-60 cells as a proof - of - concept of the technique . a limitation that the authors point out in their dac system is that in some cases , more than one cell became confined in a trap . ( a ) a pseudo - colored sem image showing a dioelectrophoresis ( dep ) trap consisting of four trapezoidaly arranged gold electrodes . this configuration induces a dipole moment in the cell in the opposite direction to the electric field . the cell is repelled from the field and stably trapped at the quadrupole s field minimum . the application of higher flow rate results in the ejection of one cell ( dark grey arrow ) from the trap leaving the other cell behind ( grey arrow ) . huang et al ( 1999 ) used dep - fff to separate cultured human breast cancer mda-435 cells from cd34 + hematopoietic stem cells . the device consisted of three layers , including a patterned layer of eight interdigitated electrodes 50 m in width and spacing , a top glass layer , and a teflon spacer in between comprising the separation chamber . a syringe pump was used to create a constant flow through the channel , forming a parabolic velocity profile . since the heights attained in the fluid channel are both a function of the cell dielectric properties and the frequency of the applied voltage , the two cell populations were individually subjected to dep - fff over a range of frequencies to obtain information about their elution characteristics as a function of frequency . these initial experiments showed that mda-435 cells took much longer to elute and even became trapped near the electrodes above 20 khz , while the cd34 + cells eluted more quickly and completely over a broader range of frequencies . the first was a trap - and - release protocol , which utilized a fixed , 40 khz frequency to trap the mda-435 cells near the electrode , while the second was a frequency sweep between 15 and 35 khz , which allowed the mda-435 cells to be levitated slightly , allowing them to flow at a lower speed down the channel . mixtures of the two cell types were then introduced into the chamber via an injection valve and allowed to equilibrate to different heights in the fluid channel for 5 minutes under an ac voltage of 10 v peak - to - peak . the cells were then subjected to either of the frequency protocols described above for 7 minutes to allow the cd34 + cells to completely elute . the frequency was then switched to 5 khz to allow the mda-435 cells to completely elute . the results showed that in both protocols , cd34 + cells were eluted with purity greater than 99% between 3 and 5 minutes , while mda-435 cells were eluted with 96% purity between 9 and 12 minutes for the trap - and - release protocol and with 99% purity for the frequency - swept protocol . these levels of purity and throughput are comparable to those of macroscale fluorescence - based and magnetic separation systems and better than that of macroscale electrophoretic systems . in another study by members of the same group ( yang et al 1999 ) , the authors found that the elution time depended on both the frequency and voltage of the ac field . larger voltages and frequencies resulted in larger dep forces and , accordingly , longer elution times . there was no significant difference in elution time between the two cell types as a function of flow rate , indicating that the height obtained within the velocity field was mainly a function of the dep forces and not hydrodynamic lift forces created by flow within the channel . in contrast to the previous study , the authors found that mda-435 cells were levitated up to 15 m higher than erythrocytes and were eluted nearly twice as fast . at a frequency of 10 khz and a flow rate of 1 ml / minute , the authors obtained mda-435 cell fractions greater than 98% in purity between 10 and 12 minutes , and erythrocyte fractions greater than 99% purity between 18 and 26 minutes . more recently , huang et al ( 2003 ) also developed a dep - based device to separate several strains of bacteria from erythrocytes , simulating the removal of biological warfare agents from blood . the separation was achieved through dep migration , since the two cell types migrated to different parts of the microelectrode structure . the device was constructed by sequential lamination of five layers : a patterned polyimide layer consisting of interdigitated dep electrodes , two pressure - sensitive acrylic adhesive layers , one of which contained microfluidic channels , a poly - carbonate substrate layer at the bottom , and a glass cover plate at the top . cell suspension samples of 5 l were loaded into the device and an ac voltage of 10 v peak - to - peak at 10 khz was applied for 5 minutes . the cells were then washed , at first with the ac voltage on and then with the voltage off , to collect the eluted samples . the authors observed that the bacteria preferentially migrated and attached to the electrodes in the presence of a dep force . in contrast , blood cells accumulated in the recessed areas between the electrodes , where field strengths were at a minimum . when the three strains of bacteria were combined and mixed with blood , the dep device was able to simultaneously separate the multiple strains from blood cells . pcr amplification of bacterial strain - specific genes did not reveal bands prior to separation , but revealed strong bands after separation , indicating that these pcr products were previously masked due to the presence of blood components . in general , higher collection efficiencies were obtained for lower initial ratios of bacteria to erythrocytes , suggesting that the separation efficiency of the device is limited by the available surface area of its electrodes to about 1 10 bacteria per run . while the above sections encompass most of the available microscale separation techniques , a number of others approaches are under development . the strategy in this approach is to lyse the undesired cell populations in a mixed suspension while leaving behind the desired cells . sethu et al ( 2004 ) have developed a microfluidic device for the separation of leukocytes from erythrocytes by lysis of the latter . this separation is usually performed by macroscale lysis using osmotic lysing agents ( such as deionized water , sodium chloride buffer , ammonium chloride sodium bicarbonate buffer ) , by density centrifugation , or some combination of macroscale lysis and centrifugation . an important characteristic of macroscale lysis that the limiting step is not the actual cell lysis ( which occurs in 2030 seconds ) , but rather the diffusion of the lysing agent in the cell suspension , which can increase the time required for complete lysis to over 5 minutes . in the device designed by sethu et al , blood cells are introduced into a channel that is also fed from either side by channels carrying lysis buffer ( figure 6 ) . the result is that the blood cells flow in a narrow stream ( 1836 m wide ) at the middle of the channel with lysis buffer on either side . this arrangement significantly reduces the time required for diffusion of the lysis buffer into the cell suspension , resulting in complete lysis within 2840 seconds , depending on the ratio of blood to lysis buffer . the reduction in the total time required for this process also has the benefits of avoiding perturbation or damage to leukocytes resulting from prolonged contact with lysing agents or from centrifugation . ( a ) a stream of blood cells is induced to flow along the center of the main channel by two adjacent streams of lysis buffer . this narrowing , shown in ( b ) , minimizes the need for lysis buffer diffusion and allows contact with the flowing blood at nearly the single cell level . sohn et al ( 2000 ) found that the capacitance of cells can be related to their dna content . using a microfluidic device fabricated from pdms and glass with embedded electrodes , the capacitance was measured across a portion of the main channel with the cells flowing past one by one . comparison of capacitance values with measurements of dna content obtained using conventional techniques yielded a linear graph with different cell types lysing at different points . for example , this technique can distinguish between mammalian erythrocytes ( which contain no dna and therefore have zero capacitance ) and leukocytes ( whose dna content , and therefore capacitance , are nonzero ) . gawad et al ( 2001 ) have described a microfluidic device that can distinguish between erythrocytes and other cells types by measurements of spectral impedance done on individual cells passing through a channel . spectral impedance of an individual cell is a function of a number of variables , including cytoplasm conductance , cell size , and membrane capacitance . this device was fabricated with polyimide and glass , and contains integrated channels and electrodes . the broad spectrum of cell separation technologies described in this review illustrates the high level of interest and activity in this area . the described size- and density - based approaches offer a great potential for separation of cell subpopulations for which specific markers are not known or can not be used ( eg , to prevent cell activation ) . affinity - based approaches ( fluorescence- , magnetic- , adhesion - based , and electrophoretic ) can be employed for fast ( ~minutes ) and continuous separation with high specificity ( ~99% ) . for all of the approaches , the design of the devices is such that they can be operated in a massively parallel fashion to increase scale and throughput without compromising purity and efficacy . furthermore , micro - fluidic separation systems can be easily incorporated with devices that perform downstream analysis such as single - cell lysis ( irimia et al 2004 ) and proteomic and genomic analysis ( huang et al 2002 ; hashimoto et al 2005 ; parano et al 2005 ; situma et al 2005 ) . given the advanced level of design , fabrication , and measurement capabilities , we expect that the focus in the coming years will shift from proof - of - concept prototypes to devices that can be economically produced and easily operated in applications such as point - of - care clinical diagnostics , drug discovery , and chemical biological agent detection .

this review describes recent work in cell separation using micro- and nanoscale technologies . these devices offer several advantages over conventional , macroscale separation systems in terms of sample volumes , low cost , portability , and potential for integration with other analytical techniques . more importantly , and in the context of modern medicine , these technologies provide tools for point - of - care diagnostics , drug discovery , and chemical or biological agent detection . this review describes work in five broad categories of cell separation based on ( 1 ) size , ( 2 ) magnetic attraction , ( 3 ) fluorescence , ( 4 ) adhesion to surfaces , and ( 5 ) new emerging technologies . the examples in each category were selected to illustrate separation principles and technical solutions as well as challenges facing this rapidly emerging field .

Introduction Size-based separation Fluorescence-based separation Magnetic separation Adhesion-based separation Electrophoretic separation Other emerging separation technologies Summary

PMC3865801

the key effector cells of the immune system , cytotoxic t lymphocytes ( ctl ) , and natural killer ( nk ) cells eliminate virus - infected and transformed cells principally through the granule exocytosis pathway . ctls and nk cells contain secretory vesicles ( granules ) that are used to store various cytotoxic proteins , including a group of proapoptotic serine proteases ( granzymes ) and perforin , a pore - forming glycoprotein . stable conjugation with a target cell results in exocytic delivery of the granule contents into the immune synapse where perforin facilitates entry of the granzymes into the target cell , triggering various apoptotic death mechanisms . the crystal structure of monomeric murine perforin has been determined to 2.75 resolution and reveals a bent and twisted four - stranded -sheet macpf domain flanked by two clusters of -helices , an epidermal growth factor ( egf ) domain , and a c - terminal c2 domain that mediates initial , calcium - dependent membrane - binding . the mechanism of pore formation has also been elucidated ; upon exposure of perforin to the neutral , calcium - rich extracellular environment of the immune synapse , deprotonation of key aspartate residues and subsequent coordination of calcium takes place . this process triggers conformational changes that result in the assembly of highly ordered oligomers of 1924 subunits that form a pore with an internal diameter of 130200 . site directed mutagenesis has established that direct ionic interactions between the opposing faces of adjacent perforin monomers ( arg213 and glu343 in particular ) serve to assist pore self - assembly and stabilization of the resulting pore . perforin is encoded on a single - copy gene in both mice and humans , and while many of the granule components possess at least some degree of redundancy , perforin is absolutely essential for protective immunosurveillance . perforin knockout mice show a reduced ability to reject many tumor xenografts , and when backcrossed with nonobese diabetic mice , the incidence of spontaneous diabetes is reduced from 77% in a perforin + /+ control to 16% in perforin - deficient mice , with onset of disease markedly delayed . this is because perforin is critical to deliver an autoimmune lethal hit against the insulin - producing pancreatic cells . perforin - deficient mice also demonstrate increased susceptibility and failure to clear many viruses and other intracellular pathogens , and highly aggressive disseminated b cell lymphomas occur in the majority of animals over the age of 12 months . in humans , complete loss of perforin function results in familial hemophagocytic lymphohistiocytosis ( fhl ) syndrome , a severe immunoregulatory disorder usually diagnosed in infancy and characterized by severe anemia and hepatosplenomegaly , fever , and thrombocytopenia . there is abundant evidence to implicate inappropriate perforin activity in a number of human pathologies , including cerebral malaria , insulin - dependent diabetes , juvenile idiopathic arthritis , and postviral myocarditis , as well as therapy - induced conditions such as allograft rejection and graft - versus - host disease . immunosuppressive agents used to treat these diseases are generally associated with a wide range of side effects , many of which arise from the unintended impact of the drug treatment on nontherapeutic targets . as perforin is expressed only by killer lymphocytes , a selective inhibitor should result in fewer off - target effects , meaning a small molecule inhibitor of perforin is of great interest as a new class of highly specific immunosuppressive agents . in our initial work in this area , we explored two series of compounds ( 1 and 2 , figure 1 ) that showed appreciable perforin - inhibitory activity but possessed limitations such as poor aqueous solubility or reduced activity in serum , precluding further development . we have substantially overcome these issues in our current series of 5-arylidene-2-thioxoimidazolidin-4-one - based inhibitors ( 3 , 4 ) and now seek to maximize inhibitory activity and optimize druglike properties . this class of rhodanine - related heterocycle has recently become the subject of considerable debate in the medicinal chemistry community because of several publications classifying such substructures as either privileged scaffolds with potentially valuable biomolecular binding properties or frequent / promiscuous hitters and pan - assay interference compounds ( pains ) which should not be pursued further . while it is clear that these chemotypes should be carefully monitored in development , recent literature contains many examples of selective inhibitors of various target proteins containing such substructures . in addition , many of these moieties also occur in drugs that are clinically useful , providing templates with valuable structural features favoring protein binding which may be exploited in an advantageous way . nevertheless , our current lead compound was subjected to and passed the pains filters ( professor jonathan baell , personal correspondence ) possibly because of the presence of an extra nitrogen in the core five - membered ring . herein we will show that we have identified an exciting new class of inhibitors that unambiguously target perforin , offering up a novel mode of action and the potential to be further developed as immunosuppressive agents for the effective treatment of transplant rejection and selected autoimmune diseases . lead compound 3 was selected from a small number of hits that showed reproducible perforin - inhibitory activity in a high throughput screen of approximately 100 000 compounds sourced from commercial libraries . compound 3 can be readily disconnected into three subunits , a 2-thioxoimidazolidin-4-one ( a ) , a furan ( b ) , and a benzofuranone ( c ) , lending itself to a study design where one unit can be independently varied in the presence of two other fixed subunits . we have reported previously our study of the structure activity relationships ( sar ) resulting from variation of the a- and b - subunits , and here we focus on the effect of introducing a wide range of c - subunits . since thiophene 4 was one of the more potent compounds in our initial work , the current study explores the effect of replacing the benzofuranone c - subunit on a fixed 2-thioxoimidazolidin-4-one / thiophene scaffold . the requirement for substitution on the c - subunit aryl ring is explored , followed by a more detailed investigation focusing on compounds containing a bicyclic moiety . the compounds of tables 1 , 2 , and 3 were prepared by heating a range of substituted aldehydes with 2-thioxoimidazolidin-4-one in acetic acid in the presence of -alanine ( schemes 15 ) . as a consequence , the majority of the chemistry that is described here involves the preparation of key aldehyde intermediates . for the compounds of table 1 , a small number of protected aldehydes ( 68 ) were prepared by suzuki reaction of 2-(5-bromothiophen-2-yl)-1,3-dioxolane ( 5 ) with substituted phenylboronic acids , followed by deprotection ( scheme 1 ) . the remaining aldehydes were commercially available with the exception of amides 3442 , which were prepared from carboxylic acid 28 using standard amide coupling conditions . table 2 contains compounds where the benzofuranone of 3 has been replaced with a variety of substituted isoindolinones ( 118135 , 165 , 166 ) and 3,4-dihydroisoquinolin-1(2h)-ones ( 167170 ) . the isoindolinone targets were prepared by bromination of methyl ester 76 , followed by ring closure onto a variety of amines to give intermediate iodides 7781 ( scheme 2 ) . these iodides were then employed in a palladium - catalyzed reaction using an agno3/kf activator system , as originally described by mori et al . coupling of the iodides with either 2-(thiophen-2-yl)-1,3-dioxolane or 2-(dimethoxymethyl)thiophene at the vacant 5-position of the thiophene gave the desired protected aldehydes 8286 . compound 82 was further modified through nah / rx alkylations of the isoindolinone nh to afford 8792 . the nme derivative 87 was alkylated a second time using lda and methyl iodide to give methylation on the isoindolinone ch2 ( 93 ) in 65% yield . deprotections of 8293 were carried out under acidic conditions to give the required aldehydes 94106 which were then reacted with 2-thioxoimidazolidin-4-one to give final compounds 118130 and 135 . likewise 136 ( table 3 ) was obtained by reaction of aldehyde 82 with imidazolidine-2,4-dione . a small set of amino - substituted compounds ( 131134 ) were also prepared from the c3 alcohol 101 . then the alcohol converted to the mesylate 108 using mesyl chloride and tea in thf . the mesylate was exchanged for iodide by heating with a large excess of nai in acetone . then the iodide of 109 was displaced with various amines to give intermediates 110113 . deprotection as described above gave the key aldehydes 114117 which were reacted with 2-thioxoimidazolidin-4-one to give final compounds 131134 . scheme 3 describes the preparation of reverse and five- and six - membered lactams 165170 ( table 2 ) . the five - membered lactam boronates 147 and 148 were prepared from the corresponding bromides 137 and 138 by pd - catalyzed reaction with bis(pinacolato)diboron . the four six - membered lactam boronates 149152 were prepared in the same manner from the bromides 143146 which were in turn synthesized from the carbamates 139142 according to literature procedures . the boronates were then each employed in a suzuki reaction with bromide 5 to give compounds 153158 which were deprotected and reacted with 2-thioxoimidazolidin-4-one to afford final compounds 165170 . for comparison with the isoindolinones , a single indole - based compound , 3-acetylindole 178 , was also prepared ( scheme 4 ) . 5-bromoindole 171 was protected and acetylated according to literature procedures , followed by displacement of the benzenesulfonyl protecting group to give the corresponding n - methylindole 174 . subsequent suzuki coupling , deprotection , and reaction with 2-thioxoimidazolidin-4-one gave target compound 178 . for the compounds of table 3 , protected bromothiophene- ( 179 ) , bromobenzene- ( 180 , 181 ) , bromopyridine- ( 182 ) , bromoindole- ( 183 , 184 ) , or bromoquinolone- ( 185 ) carboxaldehydes were reacted with either commercially available 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-1-one or boronate 149 as described above , deprotected to aldehydes 195203 , and converted to final products 204212 ( scheme 5 ) . in the first instance , the isobenzofuran-1(3h)-one c - subunit was replaced with a large range of substituted - benzene derivatives ( table 1 ) . substitution on this ring is clearly required , with phenyl ( 43 ) possessing no detectable activity . each position was then surveyed with a range of substituents , and while methyl and methoxy proved to be inactive ( see supporting information ) , a variety of electron - withdrawing substituents ( halogen , cf3 , cyano , hydroxyl , carboxyl ) did produce activity . the results for a series of chloro ( 4446 ) , fluoro ( 47 , 48 ) , and cf3 ( 50 , 51 ) compounds show a clear preference for substitution at the 4-position . carboxy - based examples ( 5961 ) were among the most potent , although in this instance the 3-carboxamide ( 60 , ic50 = 0.79 m ) was slightly better than its 4-isomer ( 61 , ic50 = 1.56 m ) . methylation of the carboxamide nh of 61 resulted in a reduction of activity ( 67 , 68 ) , while amide - linked weakly basic side chains ( 6971 ) were generally poor . the corresponding amide - linked hydroxylated side chains ( 7275 ) , however , exhibited significant activity ( ic50 = 2.444.35 m ) but were still not as potent as the primary amide 61 . other compounds with good activity were the 4-br ( 55 , 1.69 m ) , 4-sme ( 56 , 1.15 m ) , and 4-acetyl ( 57 , 1.67 m ) analogue of 4 , compound 62 , showed a 4-fold reduction in activity over its cyclic counterpart ( ic50 = 3.03 m compared to 0.78 m ) . testing was carried out over a range of concentrations , with the ic50 being equal to the concentration at which 50% inhibition of the lysis of jurkat cells by purified recombinant perforin was observed , as measured by cr release . although the above study ( table 1 ) produced some potent compounds , none were an improvement on lead compound 4 . an alternative approach where the entire isobenzofuran-1(3h)-one unit was replaced with an isoindolin-1-one was therefore implemented ( table 2 ) , resulting in a series of compounds considered less susceptible to hydrolysis ( amide versus ester ) and where the nitrogen atom provides an additional position at which a side chain can be appended . isoindolin-1-one 118 showed a reduction of activity over lactone 4 ( from ic50 = 0.78 m to ic50 = 2.55 m ) . activity was restored through substitution on the nitrogen atom , with both methyl - substituted ( 119 , ic50 = 0.51 m ) and ethyl - substituted ( 120 , ic50 = 0.60 m ) compounds more potent than the lead compound 4 . a more bulky substituent such as isopropyl ( 123 , ic50 = 4.42 m ) was less acceptable , but the acetate 128 and carbamate 129 both showed excellent activity ( ic50 = 0.93 and 0.55 m , respectively ) . in this series it also appears possible to introduce various side chains off the isoindolinone and maintain activity , with the two alcohols 125 and 126 among the most potent compounds of the class ( ic50 = 0.78 and 0.53 m ) . diol 127 and o - acetate 124 also demonstrated good activity , but when a basic amino substituent was introduced in an attempt to improve solubility , activity was virtually abolished with only morpholine ( 130 , not reproducible ) , piperidine 132 , and pyrrolidine 134 ( ic50 = 11.78 and 8.93 m ) showing any evidence of activity . introduction of a methyl substituent on the lactam ring ( 135 ) resulted in poorer activity than the corresponding unsubstituted analogue ( 135 ic50 = 3.21 m vs 119 ic50 = 0.51 m ) . since the 3-carboxamide 60 was 2-fold more potent than 4-carboxamide 61 in the substituted benzene series ( table 1 ) , the effect of reversing the orientation of the lactam ring was investigated ( 165 ) . the same result was observed ; the ic50 for the compound with the lactam amide in the 3-position 165 ( relative to the thiophene ) was around 2-fold better than the corresponding lactam amide in the 4-position ( 118 ) ( ic50 = 1.38 m compared to 2.55 m ) . expansion from a five - membered lactam ring ( 118 ) to a six - membered lactam ring ( 167 ) also resulted in a significant increase in activity ( from ic50 = 2.55 to ic50 = 1.14 m ) and improved a further 2-fold ( to 0.64 m ) through introduction of the reverse 3-lactam amide ( 168 ) . n - methylation of 165 , 167 , and 168 to give 166 , 169 , and 170 , respectively , did not result in the same increase in activity seen when 118 was alkylated to give the analogous n - methyl compound 119 . testing was carried out over a range of concentrations , with the ic50 being equal to the concentration at which 50% inhibition of the lysis of jurkat cells by purified recombinant perforin was observed , as measured by cr release . compound 178 contains a 3-acetylindole c - subunit , and its synthesis is described in scheme 4 . compounds 136 and 204212 ( table 3 ) combine those b - subunits which were previously identified as having excellent activity in the isobenzofuranone - based series , with selected isoindolinones and 3,4-dihydroisoquinolin-1(2h)-ones from the current series . compound 136 contains a imidazolidine-2,4-dione a - subunit and is the only example to show an improvement , being 3-fold better than the analogous isobenzofuranone reported in our initial paper ( ic50 = 0.40 m compared to 1.19 m ) . all other compounds ( 204212 ) contain a 2-thioxoimidazolidin-4-one a - subunit and are 1.5- to 4-fold poorer than the corresponding isobenzofuranones ( see figure 1 in supporting information ) . testing was carried out over a range of concentrations , with the ic50 being equal to the concentration at which 50% inhibition of the lysis of jurkat cells by purified recombinant perforin was observed , as measured by cr release . the 2-thioxoimidazolidin-4-one a - subunit is replaced with a imidazolidine-2,4-dione . those compounds that showed good inhibition of the purified protein were then also evaluated for their ability to inhibit perforin produced by an intact nk cell line ( khyg-1 ; see experimental section for details ) ; this assay is a better model for in vivo studies , as the perforin is delivered by a functional nk cell . to confirm that inhibition of nk cell function was due to blocking the action of perforin and not nonspecific toxicity against the effector cell , viability was also measured 24 h after the nk cells were exposed to the compounds ( 4 h , 37 c ) , then washed and replated in culture . elimination of any nonspecific toxicity is important because ctls and nk cells play a key role in the overall immunological response , meaning that any potential perforin - targeted treatment must allow rapid recovery of these cytotoxic effector cells . for the purposes of this study , compounds were classified as toxic if nk cell viability fell below 70% at 24 h. the data contained in table 4 were used to identify compounds suitable for advancement into in vivo pharmacokinetic studies , as a prelude to in vivo efficacy testing . in the absence of serum , the majority of compounds possessed similar or improved inhibitory activity compared to previous lead 4 , notably 118 , 135 , and 167 , which all showed excellent activity in this more demanding system ( 56% , 90% , 83% compared to 53% ) . as reported with previous series , a number of compounds were adversely affected in the presence of serum , precluding their further consideration ( 125 , 126 , 129 , 168 , 207 ) . the solubility of all compounds was substantially improved by their conversion to sodium salts , resulting in an enhanced drug profile , although some examples ( 118 , 119 , 129 , 136 , 168 ) still proved too insoluble in this form for administration in vivo . compounds 119 and 167 also showed some evidence of toxicity toward the nk cells ( 28% and 38% survival at 20 m , respectively ) . the compound with the best overall profile for an in vivo candidate was 135 , combining superior activity in the presence and absence of serum ( 90% and 67% , respectively ) with good viability ( 74% ) and aqueous solubility ( 1408 g / ml ) . in addition , when 167 was tested at a lower concentration of 5 m , the resultant lack of nk toxicity suggested a possible therapeutic window , meaning this compound was also selected as an in vivo candidate . inhibition by compounds ( 20 m ) of the perforin - induced lysis of k562 target cells when co - incubated with khyg-1 human nk cells ( see experimental section ) . percent inhibition calculated compared to untreated control . as for footnote a , but in the presence of 10% mouse serum . viability of khyg-1 nk cells after 24 h by trypan blue exclusion assay ( see experimental section ) . compound 167 was also tested at 5 m to test for activity under less toxic conditions . blood samples were collected at multiple time - points after dosing at 5 mg / kg in a solution of 20% hydroxypropyl--cyclodextrin by intravenous injection . both compounds were determined to have acceptable drug exposures ( auc = 1151 and 955 hng / ml , respectively ) , despite high clearance and volume of distribution values ( table 5 ) . on this basis , 135 and 167 were progressed to maximum tolerated dose ( mtd ) studies by single or multiple doses ( intraperitoneally ) to establish an appropriate level for efficacy studies . both compounds were found to be well tolerated after a single dose of 80 and 60 mg / kg , respectively , while when administered twice daily over 3 days , 60 and 40 mg / kg were found to be suitable for multiple dosing . the results were processed using a bolus iv dose and two - compartmental model with phoenix winnonlin 6.2 ( pharsight corporation , st . the derived parameters are maximum plasma concentration ( cmax ) , the area under the curve ( auc ) , plasma half - life ( t1/2 ) , volume of distribution ( v ) , and clearance ( cl ) . while several series of potent inhibitors have now been identified , little is known about the mechanism by which the cytolytic activity of perforin action is blocked . in order to find out more about the nature and degree of inhibitor binding , recombinant purified mouse perforin was immobilized on a surface plasmon resonance ( spr ) s - series cm5 sensor chip using standard amide coupling methodology . then solutions of in vivo candidates 135 and 167 in buffer were passed over the covalently bound protein , and relative binding affinities were determined using a biacore t200 spr machine . inhibitors 135 and 167 were found to bind reversibly to the immobilized perforin , and during injection a steady - state response was achieved within seconds ( figures 2 and 3 in supporting information ) . at the end of the injection the compounds dissociated rapidly with an almost immediate return to baseline resonance response . any nonspecific binding to the sensor chip surface was accounted for through the response in the reference flow cell . because of the relative insolubility of the compounds in the running buffer , low analyte concentrations were required ( 2.5 m ) and the relative resonance responses at steady state were typically below 10 response units ( ru ) . however , although the absolute response values were low , it is clear that 135 and 167 can bind to perforin even at low concentrations . these data demonstrate for the first time that these inhibitors possess the ability to bind reversibly to perforin monomers . prior to this experiment it was also not known whether immobilization of perforin monomers to the sensor chip surface via ( random ) amide couplings could render potential binding sites for inhibitors inaccessible . combined , these findings show that spr biosensor analysis can be a useful technique to determine perforin protein drug binding interactions . in order to investigate whether 167 inhibited perforin directly in the context of the physiological immune synapse , we also used real - time microscopy to analyze the functional interaction between primary human natural killer cells and their cognate model targets , hela cells . when the killer cell engages with its target cell , a cascade of downstream signaling events leads to the influx of extracellular ca ( as detected by the calcium ionophore fluo-4 ) . as shown recently , ca influx directly precedes degranulation and the release of perforin into the synaptic cleft . perforin then forms pores in the target cell membrane , which can be detected as an influx of extracellular propidium iodide ( pi ) , a dye that gains fluorescence upon binding to the cytosolic rna . calcium influx into the killer cell and pi uptake by the target cell thus serve as indicators of a functional immune synapse . in the dmso - treated control cells , every natural killer cell that engaged with its target cell ( n = 50 cells ) delivered functional perforin to the target ( figure 2 ) . by contrast , in the presence of 20 m 167 , only 60% of killer cells delivered functional perforin to the target . formation of the perforin pore was blocked in the remaining 40% of synapses , despite effective target cell engagement ( figure 2 ) . these data demonstrate that 167 directly inhibits perforin - induced lysis through reduction of cell membrane binding and/or prevention of transmembrane pore formation , thus preventing target cell death . the current study has resulted in further optimization of a novel new series of small - molecule inhibitors of the pore - forming protein perforin . by building on our previous studies , we have designed compounds that possess enhanced druglike properties compared to earlier structures . we also report new mechanistic evidence that reveals a specificity for the granule exocytosis pathway , of which perforin is an integral component . structure activity relationships for variation of the c - subunit on a 2-thioxoimidazolidin-4-one / thiophene scaffold showed a need for substitution , especially at the 4-position , for simple substituted - benzene derivatives ( table 1 ) . in this series the 3- and 4-carboxamides 60 and 61 proved to be the most potent , although this was limited to primary amides , as the introduction of n - substitution and extended hydroxyalkyl or aminoalkyl side chains ( 6775 ) resulted in a loss of activity . the acyclic analogue of the lead compound ( 62 ) also showed an almost 4-fold reduction in activity , suggesting retention of a bicyclic c - subunit to be the best approach . the isobenzofuranone of 4 was therefore replaced with a variety of isomeric isoindolinones and 3,4-dihydroisoquinolin-1(2h)-ones . n - substitution on the isoindolinone nitrogen was beneficial , with methyl ( 119 ) , ethyl ( 120 ) , hydroxyethyl ( 125 ) , and hydroxypropyl ( 126 ) substituents all demonstrating excellent activity ( ic50 = 0.51 , 0.60 , 0.78 , 0.53 m , respectively ) . comparable substitution on the 3,4-dihydroisoquinolin-1(2h)-one nitrogen did not improve activity ( comparing 167/169 and 168/170 pairings ) , although these compounds ( 167 , 168 ) still showed potent inhibitory activity in their own right . lastly , a series of hybrid compounds were prepared ( table 3 ) , combining b - subunits that were previously identified as conferring good activity with the above optimized c - subunits . in this instance , the isoindolinones were all poorer than the corresponding isobenzofuranones ( figure 1 of supporting information ) . a smaller subset of active compounds were then screened for their ability to inhibit perforin - dependent lysis induced by whole nk cells , resulting in the selection of 135 and 167 as suitable candidates for in vivo studies . problems observed previously , such as poor aqueous solubility , inactivity in serum , and toxicity have been substantially overcome in the present series . the mechanism of perforin inhibition by these compounds has also been investigated further , and for the first time , compounds with in vitro inhibitory activity have been shown to reversibly bind perforin by spr detection . target cell interaction demonstrates that the compounds block perforin within the immunological synapse and prevent target cell death but have no effect on the stability of immune synapse formation between the two cells . these inhibitors almost certainly target perforin after its release into the immune synapse , consistent with the fact that the original screen through which the inhibitors were identified featured the use of purified perforin protein rather than perforin delivered by an intact killer cell . together , the data we have amassed demonstrate that this series of compounds are selective inhibitors of perforin and not nonspecific promiscuous binders . we have previously shown that these compounds do not inhibit the mechanistically related pore - forming protein pneumolysin and that we observe a wide range of activities ( 0.36 to > 20 m ) against both isolated perforin and perforin produced by whole nk cells . furthermore , the membrane - piercing terminal components of the mammalian membrane attack complex of complement ( mac ) , which share significant structural similarity with perforin and alternative , receptor - mediated proapoptoic pathways used by cytotoxic lymphocytes , are not inhibited ( data not shown ) . these observations are recapitulated in the current report by the clear sar demonstrated by the c - subunit ( ic50 of 0.51 to > 20 m ) , irrespective of the presence of a 5-arylidene-2-thioxoimidazolidin-4-one as the a - subunit . a range of activities was also observed against perforin released by intact nk cells , a far more rigorous model that requires the compound to block perforin released into the immunological synapse that transiently forms between the killer cell and its target . our live cell imaging analyses clearly show for the first time that perforin operating within a functional immune synapse , an intricate microenvironment comprising many proteins taking part in various complex signaling pathways , can be selectively targeted to inhibit perforin - dependent apoptosis of the target cell . in summary , we have taken significant steps toward elucidating the role of perforin in the granule exocytosis pathway , using small - molecule inhibitors to investigate binding affinity and probe the mechanism of membrane interaction and subsequent pore formation . we have found compounds 135 and 167 to be both potent and soluble inhibitors of perforin in vitro . these compounds also possess appropriate in vivo pharmacokinetic characteristics and toxicity profiles for their selection as in vivo efficacy candidates in our pursuit of a novel class of perforin inhibitors suitable for the effective treatment of autoimmune - based diseases and transplant rejection . analyses were performed by the microchemical laboratory , university of otago , dunedin , new zealand . nmr spectra were measured on a bruker avance 400 spectrometer and referenced to me4si . mass spectra were recorded either on a varian vg 7070 spectrometer at nominal 5000 resolution or a finnigan mat 900q spectrometer . full experimental information for all remaining final compounds and intermediates is given in supporting information . the acetal 5 ( 278 mg , 1.18 mmol ) was then dissolved in toluene ( 12 ml ) , to which was added a suspension of 4-(hydroxymethyl)benzeneboronic acid ( 150 mg , 0.99 mmol ) in etoh ( 4 ml ) . a solution of 2 m na2co3 ( 3 ml ) and pd(dppf)cl2 ( 40 mg , 0.05 mmol ) was added , and the entire mixture was heated at reflux under nitrogen for 2 h. when the mixture was cooled , all solvents were removed under reduced pressure and the resulting residue was partitioned between water ( 50 ml ) and ch2cl2 ( 50 ml ) then the combined organic fractions were dried ( na2so4 ) , filtered , and the solvent was removed under reduced pressure to afford a residue which was purified by flash column chromatography on silica gel ( 20% etoac / hexanes as eluant ) to give 6 as a pale yellow solid ( 171 mg , 66% ) . h nmr [ 400 mhz , ( cd3)2so ] 7.60 ( d , j = 8.3 hz , 2 h ) , 7.36 ( d , j = 3.6 hz , 1 h ) , 7.35 ( d , j = 8.4 hz , 2 h ) , 7.20 ( d , j = 3.8 hz , 1 h ) , 6.04 ( s , 1 h ) , 5.19 ( t , j = 5.6 hz , 1 h ) , 4.51 ( d , j = 5.5 hz , 2 h ) , 4.104.07 ( m , 2 h ) , 3.934.00 ( m , 2 h ) . lrms ( apci ) calcd for c14h15o3s 263 ( mh ) , found 263 . this material contained 5% of deprotected aldehyde which was carried into the next step . compound 6 ( 171 mg , 0.65 mmol ) was dissolved in acetone ( 10 ml ) , to which was added 1 m hcl ( 2 ml ) . this mixture was stirred at room temperature for 6 h , then concentrated under reduced pressure to afford a pale yellow suspension which was extracted into ch2cl2 ( 2 50 ml ) . the combined organic fractions were evaporated down to give 24 as a yellow solid ( 142 mg , 100% ) . h nmr [ 400 mhz , ( cd3)2so ] 9.90 ( s , 1 h ) , 8.03 ( d , j = 3.9 hz , 1 h ) , 7.76 ( d , j = 8.3 hz , 2 h ) , 7.72 ( d , j = 4.0 hz , 1 h ) , 7.42 ( d , j = 8.4 hz , 2 h ) , 5.26 ( t , j = 5.7 hz , 1 h ) , 4.54 ( d , j = 5.6 hz , 2 h ) . lrms ( apci ) calcd for c12h11o2s 219 ( mh ) , found 219 . 5-(4-(hydroxymethyl)phenyl)thiophene-2-carbaldehyde ( 24 ) ( 140 mg , 0.64 mmol ) , 2-thioxoimidazolidin-4-one ( 89 mg , 0.77 mmol ) , and -alanine ( 69 mg , 0.77 mmol ) were suspended in acoh ( 5 ml ) , and the mixture was heated at reflux for 15 h. when the mixture was cooled , an orange solid crystallized out of solution which was collected by filtration and purified by flash column chromatography on silica gel ( 20% thf / hexanes ) , giving 58 as an orange solid ( 18% ) , mp ( thf / n - pentane ) 230234 c . h nmr [ 400 mhz , ( cd3)2so ] 12.37 ( s , 1 h ) , 11.95 ( s , 1 h ) , 7.83 ( d , j = 4.0 hz , 1 h ) , 7.72 ( d , j = 8.3 hz , 2 h ) , 7.65 ( d , j = 4.0 hz , 1 h ) , 7.44 ( d , j = 8.4 hz , 2 h ) , 6.63 ( s , 1 h ) , 5.10 ( s , 2 h ) , 2.08 ( s , 3 h ) . lrms ( apci ) calcd for c17h15n2o3s2 359 ( mh ) , found 359 . ( c17h14n2o3s2 ) c , h , n. 4-(5-formylthiophen-2-yl)benzoic acid ( 28 ) ( 85 mg , 0.37 mmol ) was dissolved in thf ( 5 ml ) , to which was added pyridine ( 289 mg , 3.67 mmol ) , followed by pentafluorophenyl trifluoroacetate ( pfp - tfa ) . this mixture was stirred at room temperature for 15 h. then all solvent was removed under reduced pressure to afford a crude solid which was dissolved in etoac ( 25 ml ) and washed with 1 m hcl ( 2 25 ml ) , water ( 25 ml ) , saturated nahco3 ( 25 ml ) , and brine ( 25 ml ) . the organic layer was then dried ( na2so4 ) , filtered and the solvent removed under reduced pressure to give the crude intermediate ester ( 146 mg , 0.37 mmol ) which was immediately dissolved in thf ( 5 ml ) and treated with 2 m methylamine in meoh ( 1.85 ml , 3.70 mmol ) . after the mixture was stirred for 1 h at room temperature , 1 m hcl ( 5 ml ) was added to hydrolyze the undesired imine . the reaction mixture was then diluted with water ( 20 ml ) and extracted with etoac ( 2 20 ml ) . then the combined organic fractions were dried ( na2so4 ) , filtered , and evaporated down to give a crude solid . h nmr [ 400 mhz , ( cd3)2so ] 9.93 ( s , 1 h ) , 8.478.53 ( br m , 1 h ) , 8.07 ( d , j = 4.0 hz , 1 h ) , 7.93 ( d , j = 8.7 hz , 2 h ) , 7.89 ( d , j = 8.7 hz , 2 h ) , 7.84 ( d , j = 4.0 hz , 1 h ) , 2.80 ( d , j = 4.5 hz , 3 h ) . lrms ( apci ) calcd for c13h12no2s 246 ( mh ) , found 246 . reaction of 34 with 2-thiohydantoin according to general procedure c gave 67 as a dark orange solid ( 76% ) , mp ( acoh ) > 300 c . h nmr [ 400 mhz , ( cd3)2so ] 12.37 ( br s , 1 h ) , 11.97 ( br s , 1 h ) , 8.47 ( br q , j = 4.5 hz , 1 h ) , 7.90 ( d , j = 8.5 hz , 2 h ) , 7.84 ( d , j = 4.0 hz , 1 h ) , 7.80 ( d , j = 8.5 hz , 2 h ) , 7.74 ( d , j = 4.0 hz , 1 h ) , 2.79 ( d , j = 4.5 hz , 3 h ) . lrms ( apci ) calcd for c16h12n3o2s2 342 ( m h ) , found 342 . ( c16h13n3o2s2 ) c , h , n. 2-thiophenecarboxaldehyde was protected as the cyclic acetal according to a literature procedure . 2-(thiophen-2-yl)-1,3-dioxolane was then reacted with 5-iodoisobenzofuran-1(3h)-one ( 77 ) using a procedure adapted from a literature reference . the iodide ( 1.23 g , 4.75 mmol ) , pdcl2(pph3)2 ( 333 mg , 0.48 mmol ) , and kf ( 1.10 g , 19.0 mmol ) were weighed into a flask and dissolved in dmso ( 35 ml ) . ( 2.22 g , 14.2 mmol ) and agno3 ( 807 mg , 4.75 mmol ) were added , . then the resulting suspension was stirred for 0.5 h at 100 c . further portions of agno3 ( 3 807 mg ) were added at 0.5 h intervals to give a total reaction time of 2 h. upon cooling , the mixture was filtered through a plug of celite which was washed well with chcl3 . the resulting chcl3 solution ( 200 ml ) was washed with water ( 3 100 ml ) , dried ( na2so4 ) , filtered and the solvent removed under reduced pressure to give a crude solid which was purified by flash column chromatography on silica gel ( 5% acetone / ch2cl2 as eluant ) . h nmr [ 400 mhz , ( cd3)2so ] 8.53 ( br s , 1 h ) , 7.85 ( d , j = 0.6 hz , 1 h ) , 7.76 ( dd , j = 7.9 , 1.6 hz , 1 h ) , 7.68 ( d , j = 7.9 hz , 1 h ) , 7.53 ( d , j = 3.7 hz , 1 h ) , 7.25 ( d , j = 3.6 hz , 1 h ) , 6.07 ( s , 1 h ) , 4.41 ( s , 2 h ) , 4.024.09 ( m , 2 h ) , 3.944.01 ( m , 2 h ) . lrms ( apci ) calcd for c15h14no3s 288 ( mh ) , found 288 . 5-(5-(1,3-dioxolan-2-yl)thiophen-2-yl)isoindolin-1-one ( 82 ) ( 150 mg , 0.52 mmol ) was dissolved in dry dmf ( 10 ml ) , and the resulting solution was cooled to 0 c ( ice / water ) . nah ( 23 mg , 0.58 mmol ) was added and the mixture stirred for 0.5 h at this temperature . methyl iodide ( 82 mg , 0.58 mmol ) was added dropwise to the solution of anion and the mixture stirred for 0.25 h at 0 c , followed by 1 h at room temperature . the mixture was diluted with water ( 50 ml ) and extracted with etoac ( 3 50 ml ) . the combined etoac fractions were washed with water ( 3 50 ml ) , brine ( 50 ml ) and dried ( na2so4 ) . filtration and removal of the solvent under reduced pressure gave a crude yellow solid which was purified by flash column chromatography on silica gel ( 5% acetone / ch2cl2 as eluant ) to give 87 as a yellow solid ( 82% ) . h nmr [ 400 mhz , ( cd3)2so ] 7.87 ( d , j = 0.7 hz , 1 h ) , 7.76 ( dd , j = 7.9 , 1.6 hz , 1 h ) , 7.67 ( d , j = 8.0 hz , 1 h ) , 7.53 ( d , j = 3.8 hz , 1 h ) , 7.25 ( d , j = 3.8 hz , 1 h ) , 6.07 ( s , 1 h ) , 4.49 ( s , 2 h ) , 4.024.09 ( m , 2 h ) , 3.943.99 ( m , 2 h ) , 3.08 ( s , 3 h ) . lrms ( apci ) calcd for c16h16no3s 302 ( mh ) , found 302 . deprotection of 87 according to general procedure b gave 95 as a yellow solid ( 89% ) . h nmr [ 400 mhz , ( cd3)2so ] 9.94 ( s , 1 h ) , 8.08 ( d , j = 4.0 hz , 1 h ) , 8.04 ( d , j = 0.8 hz , 1 h ) , 7.91 ( dd , j = 7.9 , 1.6 hz , 1 h ) , 7.86 ( d , j = 4.0 hz , 1 h ) , 7.74 ( d , j = 7.9 hz , 1 h ) , 4.53 ( s , 2 h ) , 3.09 ( s , 3 h ) . lrms ( apci ) calcd for c14h12no2s 258 ( mh ) , found 258 . reaction of 95 with 2-thiohydantoin according to general procedure c gave 119 as an orange solid ( 92% ) , mp ( acoh ) > 300 c . h nmr [ 400 mhz , ( cd3)2so ; observe e- and z- isomers separately ) ] 11.9012.38 ( m , 2 h ) , 7.92 ( br d , j = 0.7 hz , 1 h ) , 7.82 ( br d , j = 4.0 hz , 0.8 h ) , 7.807.84 ( m , 1 h ) , 7.757.78 ( m , 1 h ) , 7.677.73 ( m , 1.2 h ) , 6.82 ( s , 0.2 h ) , 6.65 ( s , 0.8 h ) , 4.51 ( s , 2 h ) , 3.09 ( s , 3 h ) . lrms ( apci ) calcd for c17h12n3o2s2 354 ( m h ) , found 354 . methyl 4-iodo-2-methylbenzoate ( 76 ) ( 860 mg , 3.12 mmol ) was dissolved in benzene ( 10 ml ) , to which were added n - bromosuccinimide ( nbs ) ( 666 mg , 3.74 mmol ) and 2,2-azobis(2-methylpropionitrile ) ( 51 mg , 0.31 mmol ) . this mixture was heated at reflux temperature for 6 h. the mixture was allowed to cool , filtered and the resulting filtrate diluted with et2o ( 100 ml ) . this solution was washed with saturated sodium metabisufite ( 50 ml ) and brine ( 50 ml ) , dried ( na2so4 ) , and filtered . removal of the solvent under reduced pressure gave a transparent oil which was purified by filtration through a plug of silica gel ( 5% etoac / hexanes as eluant ) . the resulting oil solidified under high vacuum to afford a white solid ( 1.08 g ) , shown to be 93% desired monobromide and 7% unreacted starting material by h nmr . this solid was used directly in the next step . the bromide ( 1.08 mg , 3.04 mmol ) was dissolved in thf ( 10 ml ) , and 2-aminoethanol ( 928 mg , 15.2 mmol ) was added . the reaction mixture was stirred at room temperature for 15 h. then all solvent was removed under reduced pressure . the resulting residue was purified by flash column chromatography on silica gel ( 10% acetone / ch2cl2 as eluant ) to give 78 as a crystalline white solid ( 562 mg , 59% over two steps ) . h nmr [ 400 mhz , ( cd3)2so ] 8.02 ( d , j = 0.7 hz , 1 h ) , 7.84 ( dd , j = 7.9 , 1.4 hz , 1 h ) , 7.45 ( d , j = 7.9 hz , 1 h ) , 4.82 ( t , j = 5.4 hz , 1 h ) , 4.52 ( s , 2 h ) , 3.583.64 ( m , 2 h ) , 3.323.57 ( m , 2 h ) . lrms ( apci ) calcd for c10h11ino2 304 ( mh ) , found 304 . 2-thiophenecarboxaldehyde was protected as the dimethyl acetal according to a literature procedure , then reacted with 78 according to general procedure e to give 83 . compound 83 ( 430 mg , 1.29 mmol ) was dissolved in acetone ( 32 ml ) , to which were added water ( 8 ml ) and p - toluenesulfonic acid ( 100 mg ) . this mixture was stirred at 50 c for 5 h. upon cooling , the reaction mixture was diluted with water ( 100 ml ) and extracted with ch2cl2 ( 3 50 ml ) . purification by flash column chromatography on silica gel ( 5% meoh / ch2cl2 as eluant ) gave 100 as a yellow solid ( 378 mg , 71% over two steps ) . h nmr [ 400 mhz , ( cd3)2so ] 9.94 ( s , 1 h ) , 8.08 ( d , j = 4.0 hz , 1 h ) , 8.04 ( d , j = 0.8 hz , 1 h ) , 7.92 ( dd , j = 7.9 , 1.6 hz , 1 h ) , 7.85 ( d , j = 4.0 hz , 1 h ) , 7.75 ( d , j = 8.0 hz , 1 h ) , 4.83 ( t , j = 5.4 hz , 1 h ) , 4.62 ( s , 2 h ) , 3.563.67 ( m , 4 h ) . reaction of 100 with 2-thiohydantoin according to general procedure c gave 124 as a red powder ( 57% ) , mp ( acoh ) 261264 c . h nmr [ 400 mhz , ( cd3)2so ] 12.39 ( s , 1 h ) , 11.98 ( s , 1 h ) , 7.927.96 ( m , 1 h ) , 7.87 ( d , j = 4.0 hz , 1 h ) , 7.84 ( dd , j = 8.0 , 1.5 hz , 1 h ) , 7.77 ( d , j = 4.0 hz , 1 h ) , 7.73 ( d , j = 7.9 hz , 1 h ) , 6.65 ( s , 1 h ) , 4.59 ( s , 2 h ) , 4.26 ( t , j = 5.4 , hz , 2 h ) , 3.78 ( t , j = 5.3 , hz , 2 h ) , 2.00 ( s , 3 h ) . hydrolysis of 124 ( 161 mg , 0.42 mmol ) was carried out by removing the previous reaction solvent ( acoh , 4 ml ) from the reaction mixture under reduced pressure and treating the crude o - acetate with k2co3 ( 289 mg , 2.09 mmol ) in a mixture of meoh ( 10 ml ) and water ( 2 ml ) at room temperature for 1 h. the meoh was removed under reduced pressure to give an orange - black oil which was cooled to 0 c ( ice / water ) and acidified with 1 m hcl . the resulting solid was collected by filtration , allowed to air - dry , then stirred in warm meoh until a homogeneous suspension was obtained . this was collected by filtration and dried under vacuum to give 125 as an orange solid ( 78 mg , 48% over two steps ) , mp ( acoh ) 285 c ( dec ) . h nmr [ 400 mhz , ( cd3)2so ; observe e- and z- isomers separately ] 11.9812.39 ( m , 2 h ) , 7.93 ( br s , 1 h ) , 7.87 ( d , j = 4.0 hz , 1 h ) , 7.83 ( dd , j = 8.0 , 1.5 hz , 0.9 h ) , 7.77 ( d , j = 4.0 hz , 1 h ) , 7.72 ( d , j = 8.0 hz , 1 h ) , 7.68 ( d , j = 4.0 hz , 0.1 h ) , 6.84 ( s , 0.1 h ) , 6.65 ( s , 0.9 h ) , 4.82 ( t , j = 5.2 hz , 1 h ) , 4.60 ( s , 2 h ) , 3.623.67 ( m , 2 h ) , 3.563.61 ( m , 2 h ) . lrms ( apci ) calcd for c18h14n3o3s2 384 ( m h ) , found 384 . in this case ( and others where the target compound contained an amine ) the acoh solvent was removed under reduced pressure to afford an orange - black oil which gave an orange solid when suspended and stirred in a mixture of acetone ( 5 ml ) and saturated nahco3 ( 10 ml ) . this solid was collected by filtration , dried under vacuum , and triturated with meoh to afford 130 as an orange solid ( 72% ) , mp ( meoh ) 261264 c . h nmr [ 400 mhz , ( cd3)2so ; observe e- and z- isomers separately ] 11.9712.29 ( m , 2 h ) , 7.94 ( s , 1 h ) , 7.807.87 ( m , 2 h ) , 7.757.79 ( m , 1 h ) , 7.72 ( d , j = 8.0 hz , 0.85 h ) , 7.68 ( d , j = 3.9 hz , 0.15 h ) , 6.84 ( s , 0.15 h ) , 6.64 ( s , 0.15 h ) , 4.61 ( s , 2 h ) , 3.66 ( t , j = 6.2 hz , 2 h ) , 3.55 ( t , j = 4.5 hz , 4 h ) , 2.57 ( t , j = 6.2 hz , 2 h ) , 2.43 ( br s , 4 h ) . lrms ( apci ) calcd for c22h23n4o3s2 455 ( m + h ) , found 455 . hrms ( esi ) calcd for c22h23n4o3s2 455.1206 ( mh ) , found 455.1201 . compound 101 ( 1.20 g , 3.98 mmol ) was dissolved in a mixture of meoh ( 50 ml ) and trimethyl orthoformate ( 5 ml ) . p - toluenesulfonic acid ( 100 mg ) was added , along with 4 molecular sieves ( 5.0 g ) . then the reaction mixture was heated at reflux for 15 h. upon cooling , the reaction mixture was diluted with ch2cl2 ( 100 ml ) and filtered through celite which was washed well with ch2cl2 . the solvent was removed from the combined filtrate under reduced pressure to give an oil which was dissolved in etoac ( 150 ml ) and washed with saturated nahco3 ( 2 100 ml ) , water ( 100 ml ) , and brine ( 100 ml ) . the organic phase was dried ( na2so4 ) , filtered and the solvent removed to afford 107 as a waxy , pale yellow solid ( 100% ) . h nmr [ 400 mhz , ( cd3)2so ] 7.86 ( d , j = 0.8 hz , 1 h ) , 7.75 ( dd , j = 8.0 , 1.5 hz , 1 h ) , 7.67 ( d , j = 7.9 hz , 1 h ) , 7.54 ( d , j = 3.7 hz , 1 h ) , 7.02 ( dd , j = 3.8 , 0.8 hz , 1 h ) , 5.66 ( d , j = 0.7 hz , 1 h ) , 4.51 ( s , 2 h ) , 4.49 ( t , j = 5.1 hz , 1 h ) , 3.57 ( t , j = 7.2 hz , 2 h ) , 3.45 ( q , j = 5.9 hz , 2 h ) , 3.32 ( s , 6 h ) , 1.76 ( pentet , j = 6.7 hz , 2 h ) . lrms ( apci ) calcd for c18h22no4s 348 ( mh ) , found 348 . alcohol 107 ( 1.29 g , 3.72 mmol ) was dissolved in dry thf ( 40 ml ) . triethylamine ( 3.76 g , 37.2 mmol ) and methanesulfonyl chloride ( 1.70 g , 14.9 mmol ) were added , and the reaction mixture was stirred at room temperature for 3.5 h. the mixture was diluted with etoac ( 200 ml ) , which was washed with water ( 100 ml ) , saturated nahco3 ( 100 ml ) , and brine ( 100 ml ) . the organic layer was dried ( na2so4 ) , filtered and the solvent removed under reduced pressure to give a residue which was purified by flash column chromatography on silica gel ( 5% acetone / ch2cl2 as eluent ) , giving 108 as a waxy yellow solid ( 1.58 g , 100% ) . h nmr [ 400 mhz , ( cd3)2so ] 7.87 ( d , j = 0.7 hz , 1 h ) , 7.76 ( dd , j = 7.9 , 1.5 hz , 1 h ) , 7.69 ( d , j = 7.6 hz , 1 h ) , 7.55 ( d , j = 3.7 hz , 1 h ) , 7.12 ( dd , j = 3.7 , 0.8 hz , 1 h ) , 5.66 ( d , j = 0.6 hz , 1 h ) , 4.53 ( s , 2 h ) , 4.25 ( t , j = 6.2 hz , 2 h ) , 3.63 ( t , j = 6.9 hz , 2 h ) , 3.32 ( s , 6 h ) , 3.18 ( s , 3 h ) , 2.04 ( pentet , j = 6.6 hz , 2 h ) . lrms ( apci ) calcd for c19h24no6s2 426 ( mh ) , found 426 . mesylate 108 ( 624 mg , 1.47 mmol ) was dissolved in acetone ( 30 ml ) at 70 c . stirring was continued at this temperature for 1 h. then the mixture was allowed to cool and was filtered through celite which was washed well with acetone . the solvent was removed from the combined filtrate under reduced pressure to give a residue which was purified by flash column chromatography on silica gel ( 10% acetone / ch2cl2 as eluant ) , giving 109 as a waxy yellow solid ( 660 mg , 96% ) . h nmr [ 400 mhz , ( cd3)2so ] 7.86 ( d , j = 0.8 hz , 1 h ) , 7.76 ( dd , j = 7.9 , 1.6 hz , 1 h ) , 7.68 ( d , j = 7.9 hz , 1 h ) , 7.55 ( d , j = 3.7 hz , 1 h ) , 7.12 ( dd , j = 3.7 , 0.8 hz , 1 h ) , 5.66 ( d , j = 0.7 hz , 1 h ) , 4.53 ( s , 2 h ) , 3.58 ( t , j = 6.9 hz , 2 h ) , 3.32 ( s , 6 h ) , 3.25 ( t , j = 6.9 hz , 2 h ) , 2.14 ( pentet , j = 6.9 hz , 2 h ) . lrms ( apci ) calcd for c18h21ino3s 458 ( mh ) , found 458 . iodide 109 ( 330 mg , 0.71 mmol ) was dissolved in dimethylacetamide ( 10 ml ) , to which was added dimethylamine ( 3.53 ml of a 2 m solution in thf ) , and the resulting mixture was stirred for 15 h at room temperature . all solvent was removed under reduced pressure to give a viscous oil which was dissolved in etoac ( 100 ml ) . this solution was washed with water ( 3 100 ml ) , brine ( 100 ml ) and dried ( na2so4 ) . filtration and removal of the solvent under reduced pressure gave the crude dimethyl acetal protected product ( 110 ) as a yellow oil . this product was then deprotected directly to the desired aldehyde according to general procedure h. trituration with et2o gave 114 as a yellow solid ( 140 mg , 60% ) . h nmr [ 400 mhz , ( cd3)2so ] 9.94 ( s , 1 h ) , 8.08 ( d , j = 4.0 hz , 1 h ) , 8.03 ( d , j = 0.7 hz , 1 h ) , 7.92 ( dd , j = 8.0 , 1.6 hz , 1 h ) , 7.86 ( d , j = 4.0 hz , 1 h ) , 7.75 ( d , j = 7.9 hz , 1 h ) , 4.55 ( s , 2 h ) , 3.56 ( t , j = 7.2 hz , 2 h ) , 2.24 ( t , j = 7.1 hz , 2 h ) , 2.13 ( s , 6 h ) , 1.74 ( pentet , j = 7.2 hz , 2 h ) . lrms ( apci ) calcd for c18h21n2o2s 329 ( mh ) , found 329 . reaction of 114 with 2-thiohydantoin according to general procedure c , followed by isolation according to general procedure j , gave 131 as a dark orange solid ( 51% ) , mp ( meoh ) 212 c ( dec ) . h nmr [ 400 mhz , ( cd3)2so ] 11.47 ( v br s , 2 h ) , 7.91 ( br s , 1 h ) , 7.82 ( dd , j = 8.0 , 1.4 hz , 1 h ) , 7.677.73 ( m , 3 h ) , 6.52 ( s , 1 h ) , 4.53 ( s , 2 h ) , 3.56 ( t , j = 7.0 hz , 2 h ) , 2.402.47 ( m , 2 h ) , 2.33 ( s , 6 h ) , 1.81 ( pentet , j = 7.2 hz , 2 h ) . lrms ( apci ) calcd for c21h23n4o2s2 427 ( mh ) , found 427 . compound 87 was dissolved in dry thf ( 10 ml ) and cooled to 78 c under n2 . lithium diisopropylamide in cyclohexane ( 0.40 ml of a 1.5 m solution , 0.59 mmol ) was added dropwise . methyl iodide ( 84 mg , 0.59 mmol ) was added dropwise with stirring continued at 78 c for 0.5 h , at which point the mixture was allowed to warm to room temperature . the mixture was diluted with saturated nh4cl ( 50 ml ) and extracted with etoac ( 3 50 ml ) . the combined etoac fractions were washed with brine ( 50 ml ) and dried ( na2so4 ) . filtration and removal of the solvent under reduced pressure gave a brown oil which was purified by flash column chromatography on silica gel ( 5% acetone / ch2cl2 as eluant ) to give 93 as a yellow oil ( 65% ) . h nmr [ 400 mhz , ( cd3)2so ] 7.92 ( t , j = 0.7 hz , 1 h ) , 7.75 ( dd , j = 7.9 , 1.4 hz , 1 h ) , 7.66 ( d , j = 7.9 hz , 1 h ) , 7.55 ( d , j = 3.7 hz , 1 h ) , 7.26 ( d , j = 3.7 hz , 1 h ) , 6.07 ( s , 1 h ) , 4.59 ( q , j = 6.7 hz , 1 h ) , 4.024.09 ( m , 2 h ) , 3.944.02 ( m , 2 h ) , 3.01 ( s , 3 h ) , 1.47 ( d , j = 6.7 hz , 3 h ) . lrms ( apci ) calcd for c17h18no3s 316 ( m + h ) , found 316 . deprotection of 93 was carried out according to general procedure b to give 106 as a beige solid ( 96% ) . h nmr [ 400 mhz , ( cd3)2so ] 9.94 ( s , 1 h ) , 8.078.10 ( m , 2 h ) , 7.91 ( dd , j = 8.0 , 1.4 hz , 1 h ) , 7.88 ( d , j = 4.0 hz , 1 h ) , 7.73 ( d , j = 7.9 hz , 1 h ) , 4.63 ( q , j = 6.7 hz , 1 h ) , 3.02 ( s , 3 h ) , 1.49 ( d , j = 6.7 hz , 3 h ) . lrms ( apci ) calcd for c15h14no2s 272 ( m + h ) , found 272 . reaction of 106 with 2-thiohydantoin according to general procedure c gave 135 as an orange solid ( 57% ) , mp ( acoh ) 286 c ( dec ) . z- isomers separately ] 11.9212.38 ( m , 2 h ) , 7.957.99 ( m , 1 h ) , 7.87 ( d , j = 4.0 hz , 0.75 h ) , 7.727.82 ( m , 2 h ) , 7.687.72 ( m , 1.25 h ) , 6.84 ( s , 0.25 h ) , 6.64 ( s , 0.75 h ) , 4.61 ( q , j = 6.7 hz , 1 h ) , 3.02 ( s , 3 h ) , 1.48 ( d , j = 6.7 hz , 3 h ) . hrms ( esi ) calcd for c18h14n3o2s2 368.0533 ( m h ) , found 368.0526 . 6-bromoisoindolin-1-one ( 137 ) was alkylated with methyl iodide and nah according to general procedure f to give 138 as a pale yellow solid ( 68% ) . h nmr [ 400 mhz , ( cd3)2so ] 7.747.79 ( m , 2 h ) , 7.56 ( dd , j = 7.9 , 0.7 hz , 1 h ) , 4.44 ( s , 2 h ) , 3.07 ( s , 3 h ) . lrms ( apci ) calcd for c9h9brno 226 , 228 ( mh ) , found 226 , 228 . 6-bromo-2-methylisoindolin-1-one ( 138 ) ( 723 mg , 3.41 mmol ) , bis(pinacolato)diboron ( 1.04 g , 4.09 mmol ) , potassium acetate ( 1.00 g , 10.2 mmol ) , and pd(dppf)cl2 catalyst ( 139 mg , 0.17 mmol ) were weighed into a flask that was sealed under n2 . dmso ( 15 ml ) was added , and the entire mixture was stirred at 90 c for 5 h. upon cooling , the reaction mixture was diluted with water ( 250 ml ) and extracted with ch2cl2 ( 5 50 ml ) . the combined ch2cl2 fractions were in turn washed with water ( 2 100 ml ) , brine ( 100 ml ) , dried ( na2so4 ) , filtered and the solvent was removed under reduced pressure to yield the crude product . purification was carried out by flash column chromatography on silica gel ( 20% thf / ch2cl2 as eluant ) to give 148 as a crystalline beige solid ( 262 mg , 28% ) . h nmr [ 400 mhz , ( cd3)2so ] 7.91 ( br s , 1 h ) , 7.85 ( dd , j = 7.6 , 1.0 hz , 1 h ) , 7.59 ( dd , j = 7.5 , 0.6 hz , 1 h ) , 4.49 ( s , 2 h ) , 3.07 ( s , 3 h ) , 1.32 ( s , 12 h ) . lrms ( apci ) calcd for c15h21bno3 274 ( mh ) , found 274 . reaction of 148 with 2-(5-bromothiophen-2-yl)-1,3-dioxolane ( 5 ) according to general procedure a followed by deprotection of 154 directly to the aldehyde according to general procedure b gave 160 as a pale yellow solid ( 91% ) . h nmr [ 400 mhz , ( cd3)2so ] 9.93 ( s , 1 h ) , 8.06 ( d , j = 4.0 hz , 1 h ) , 7.998.03 ( m , 2 h ) , 7.88 ( d , j = 4.0 hz , 1 h ) , 7.70 ( dd , j = 7.8 , 0.8 hz , 1 h ) , 4.52 ( s , 2 h ) , 3.10 ( s , 3 h ) . lrms ( apci ) calcd for c14h12no2s 258 ( mh ) , found 258 . reaction of 160 with 2-thiohydantoin according to general procedure c gave 166 as an orange solid ( 89% ) , mp ( acoh ) > 310 c . h nmr [ 400 mhz , ( cd3)2so ; observe e- and z - isomers separately ] 11.9712.34 ( m , 2 h ) , 7.917.96 ( m , 2 h ) , 7.82 ( d , j = 4.0 hz , 0.85 h ) , 7.757.78 ( m , 1 h ) , 7.69 ( d , j = 4.0 hz , 0.15 h ) , 7.66 ( d , j = 8.6 hz , 1 h ) , 6.84 ( s , 0.15 h ) , 6.66 ( s , 0.85 h ) , 4.40 ( s , 2 h ) , 3.10 ( s , 3 h ) . lrms ( apci ) calcd for c17h12n3o3s2 354 ( m h ) , found 354 . as reported previously , compound 3 was identified from screening a commercial library of 100 000 compounds for the ability to reproducibly inhibit perforin - mediated lysis of srbc at a compound concentration of 100 m . the ability of the compounds to inhibit the lysis of nucleated ( jurkat t lymphoma ) cells in the presence of 0.1% bsa was measured by release of cr label . jurkat target cells were labeled by incubation in medium with 100 ci cr for 1 h. the cells were then washed three times to remove unincorporated isotope and resuspended at 1 10 cells per milliliter in rpmi buffer supplemented with 0.1% bsa . each test compound was preincubated to concentrations of 20 , 10 , 5 , 2.5 , and 1.25 m with recombinant perforin for 30 min with dmso as a negative control . cr labeled jurkat cells were then added , and the cells were incubated at 37 c for 4 h. the supernatant was collected and assessed for its radioactive content on a counter ( wallac wizard 1470 automatic counter ) . each data point was performed in triplicate , and an ic50 was calculated from the range of concentrations described above . compounds with ic50 < 1 m were titrated to lower concentrations in the same manner as above , to determine an accurate ic50 . khyg-1 cells were washed and resuspended in rpmi + 0.1% bsa at 4 10 cells / ml , and an amount of 50 l of khyg-1 cells was dispensed to each well of a 96-well v - bottom plate . rpmi ( 50 l ) + 0.1% bsa or 10% ( final concentration ) of serum was added to each well . then test compounds were added to khyg-1 cells at various concentrations up to 20 m and incubated at room temperature for 20 min . 1 10 k562 target cells were labeled with 75 ci cr in 200 l of rpmi for 90 min at 37 c . cells were washed as described above and resuspended in 5 ml of rpmi + 0.1% bsa . an amount of 50 l of cr labeled k562 leukemia target cells was added to each well of the khyg-1 plate ( effector / target 2:1 ) and incubated at 37 c for 4 h. cr release was assayed using a skatron harvesting press and radioactivity estimated on a wallac wizard 1470 automatic counter ( turku , finland ) . the percentage of specific cytotoxicity was calculated using the following formula : and expressed as the mean of triplicate assays standard error of the mean . the toxicity assay was carried out in exactly the same manner as the killing assay above , but instead of adding the labeled k562 target cells , 100 l of rpmi 0.1% bsa was added . cells were incubated for 4 h at 37 c and then washed 3 in rpmi + 0.1% bsa . cells were then resuspended in 200 l of complete medium and incubated for 1824 h at 37 c . viable ( clear ) cells and total ( clear + blue ) cells were counted , and the percentage of viable cells was calculated compared to dmso treated cell control ( % viability ) . pharmacokinetic studies were carried out in healthy cd-1 male mice ( n = 3 at each time point ) using intravenous administration of 135 and 167 at a concentration of 5 mg / kg in 20% 2-hydroxypropyl--cyclodextrin ( sigma - aldrich ) . blood was collected by cardiac puncture at 5 and 30 min and 1 , 2 , 4 , and 6 h postdose in ice - cold k2-edta tubes . plasma samples were separated by centrifuging at 4000 g for 8 min and stored at 80 c until analysis . then the sample ( 10 l ) was transferred to a clean microcentrifuge tube and mixed with three volumes of ice - cold acetonitrile / methanol ( 3:1 v / v ) [ containing 213 as internal standard ( table 6 , supporting information ) ] to precipitate the plasma proteins . the tubes were centrifuged at 15000 g for 10 min to obtain the clear supernatant which was mixed with 0.01% formic acid water ( 1:1 ) , and concentration of the parent drug was measured through injection of 1020 l into an lc ms / ms ( agilent 6410 series triple quadrupole mass spectrometry detector system ) . a zorbax sb - c18 , 50 mm 2.1 mm 5 m column was used at a 0.5 ml / min flow rate , and elution was with a gradient of organic phase ( a ) , acetonitrile in water ( 80:20 v / v ) containing 0.01% formic acid and aqueous phase parent drug was quantified against a standard curve 0250 ng / ml in control mouse plasma . plasma samples from 5 min , 30 min , and 1 h were diluted with control mouse plasma ( 1:20 for 5 min , 1:10 for 30 min and 1 h samples ) to fit within this range . the resultant concentration vs time data were fitted ( using phoenix winnonlin 6.2 ; pharsight corporation , st . louis , mo ) to calculate pharmacokinetic parameters including half - life ( t1/2 ) , maximum concentration ( cmax ) , area under the curve ( auc0 ) , volume of distribution ( v ) , and clearance ( cl ) . 3 intraperitoneal administration through a classical dose escalation design in c57bl/6 mice and defined as a dose level that was tolerated by mice ( n = 23 mice / group ) with a body weight loss of < 10% and no death or detectable sickness . all animal experiments were approved by the animal ethics committee of the university of auckland . binding experiments with surface immobilized wild - type perforin monomers on a s - series cm5 sensor chip ( ge healthcare life sciences ) were carried out using a biacore t200 spr machine ( ge healthcare life sciences ) . perforin monomer solution ( 25 g / ml in 10 mm sodium acetate , ph 5.5 ) was injected over the sensor chip surface under standard amide coupling conditions to obtain covalent binding of the protein ( density = 4257 ru ) . the initial and running buffers were made from a 10 stock solution of hepes buffered saline ( hbs ) . 10 hbs ( 50 ml ) was diluted to 500 ml with water and filtered through a 0.22 m filter ( initial buffer ) . this initial buffer ( 380 ml ) was added to dmso ( 20 ml ) to prepare the 5% dmso running buffer . these solutions were diluted in hepes initial buffer to 5% dmso ( 125 m ) and then subsequently diluted to the required concentrations using hepes running buffer containing 5% dmso to maintain a constant dmso concentration . a solvent correction was run before and after the injections to account for differences in dmso concentrations between buffer and analyte solutions . time - lapse microscopy and image analysis were performed as described previously . in brief , primary human natural killer cells were isolated from the peripheral blood of four unrelated donors using negative selection , as described in the above reference . natural killer cells were labeled with 1 m fluo-4am for 20 min at 37 c/5% co2 , and then cells were washed and preincubated for 10 min in rpmi culture medium containing 0.2% ( v / v ) dmso or 20 m 167/0.2% ( v / v ) dmso at room temperature before combining with hela cell targets preseeded in chamber slides . the final mixture contained complete rpmi-1640 culture medium supplemented with 100 m propidium iodide , 0.2% ( v / v ) dmso or 20 m 167/0.2% ( v / v ) dmso .

a series of novel 5-arylidene-2-thioxoimidazolidin-4-ones were investigated as inhibitors of the lymphocyte - expressed pore - forming protein perforin . structure activity relationships were explored through variation of an isoindolinone or 3,4-dihydroisoquinolinone subunit on a fixed 2-thioxoimidazolidin-4-one / thiophene core . the ability of the resulting compounds to inhibit the lytic activity of both isolated perforin protein and perforin delivered in situ by natural killer cells was determined . a number of compounds showed excellent activity at concentrations that were nontoxic to the killer cells , and several were a significant improvement on previous classes of inhibitors , being substantially more potent and soluble . representative examples showed rapid and reversible binding to immobilized mouse perforin at low concentrations ( 2.5 m ) by surface plasmon resonance and prevented formation of perforin pores in target cells despite effective target cell engagement , as determined by calcium influx studies . mouse pk studies of two analogues showed t1/2 values of 1.11.2 h ( dose of 5 mg / kg iv ) and mtds of 6080 mg / kg ( ip ) .

Introduction Results and Discussion Conclusions Experimental Section

PMC2651738

the lipid bilayer plays critical roles in the biochemistry of cells , the most basic of which being its role in defining the shape of the cell and organelles . the unique arrangement of hydrophilic and hydrophobic groups at the membrane exterior and interior regions , respectively , allows for a selective inhibition of foreign agents and transport of essential molecules . as a consequence , the functional versatility of the cell membrane is also in tune with its highly inhomogeneous composition and structure , wherein receptors , channels , peptides , and other molecules create a vast chemical mixture and diverse geometry.(5 ) the thermodynamics of adhesion and membrane insertion of biomolecules is therefore the subject of much research . in the context of protein folding , for instance , understanding how membrane proteins insert and simultaneously fold to assume their functional three - dimensional structure is of great interest.(6 ) this poorly understood process can involve the cooperative action of several amino acid residues that work in concert toward both membrane penetration and organization to a particular shape . researchers are also studying how smaller biomolecules insert into the membrane and aggregate , and to what extent these processes affect the physical properties of the membrane . several experimental methods have been employed to describe the equilibrium properties of peptide insertion , that is , to describe the initial and final states and the associated thermodynamic properties . the most important thermodynamic parameter in this context is the free energy change , g , defined here to be the energetic change upon the transfer of the peptide from its initial aqueous environment to its final state in membrane ; g < 0 indicates a favorable insertion process . studied the free energy of amino acid partitioning first in an octanol / water(12 ) membrane mimic and later in a synthetic membrane.(13 ) in the latter study , they derived a thermodynamic scale for the partitioning of small model peptides between membrane and water . by altering a single residue in the test peptide , the contribution of each of the 20 amino acids to the transfer free energy for instance , in the case of the hydrophobic amino acid leucine , the free energy of transfer is 0.56 0.04 kcal mol . also contemplated how peptide aggregation or formation of higher order molecular assemblies might occur in membranes . concurrently , computational methods have played significant roles in elucidating the atomic - level details that are not always accessible by experiment . the advance of computational power and the development of faster algorithms enables modeling of increasingly larger membrane systems . for example , aliste and tieleman used md simulations to study the partitioning of the wimley and white peptides in a model membrane , and the results provided the details of the peptidemembrane atomic interactions responsible for insertion.(21 ) the partitioning of the various amino acids into a model membrane host has also been studied.(22 ) a unique benefit of computation is its ability to shed light on the free energy of peptidemembrane binding , as well as to characterize the energetic landscape or the pathways of insertion and aggregation . one such study examined the free energy profile ( or the potential of mean force , pmf ) of the chromophore indole ring as it traveled through the membrane.(23 ) in this work , the authors explored how different parametrizations of the indole moiety can affect the free energy calculations ; they also pinpointed some key properties of the reaction coordinate , including the locations of energetic barriers . in this study , we employ a molecular dynamics ( md ) technique known as umbrella sampling to simulate the insertion of a model hydrophobic peptide into a membrane . the model of choice is the small hexapeptide consisting of one tryptophan ( trp ) and five leucine residues ( wl5 ) , the same model system used in the experiments of wimley and white.(13 ) we computed the pmf as the peptide moves from the solvent and inserts into and completely traverses across the membrane . to our knowledge , this is the first attempt in computing the free energy profile of a full length peptide as it crosses a membrane . we analyzed the dynamical and structural properties of the peptide during this process . by coupling the computed free energy profile with the experimental results of wimley and white , we propose a thermodynamic model for the insertion and aggregation of hydrophobic peptides in a membrane host . each leaflet of the membrane can be divided into three regions based on the polarity of the constituent atoms : the solventheadgroup interface ( s / hg ) , where the first two solvent shells ( 6 ) merge with the choline and phosphate head groups of the phospholipids ; the glycerol or headgroupcore interface ( hg / c ) , where the lipid head groups mix with the hydrophobic fatty acid chains ; and the core , the region occupied by the aliphatic lipid tails . by using these three regions as landmarks , the free energy profile of the peptide wl5 as it traverses across the membrane see supporting information for a figure ( s1 ) demonstrating the extent of convergence of this profile ( in the allotted sampling time ) . the profile exhibits minima at both the s / hg and the hg / c interfaces of each leaflet , the latter being the global minimum . note that two peptide orientations ( one per leaflet ) were used to determine whether the initial orientation affects the energetics of insertion . the dashed line in figure 1 clearly shows that the initial orientation does indeed affect the profile . such an asymmetric profile implies that even for a rudimentary peptide such as wl5 , there may be more than one possible path of membrane insertion . free energy profile ( symmetric - heavy blue line , asymmetric - dashed line ) of the peptide as a function of position along the z dimension ( negative / positive values correspond to the lower / upper leaflets , respectively ) . pink marks the regions of the solvent / lipid headgroup interface ( s / hg in the lower leaflet , hg / s in the upper ) . beige indicates the headgroup / core interface ( hg / c in the lower leaflet , c / hg in the upper ) . the conformation of the peptide , as judged by the distribution of / angles , assumes that of a strand , especially when the peptide is positioned somewhere in the membrane ; see figure s2 of supporting information . most noticeably , the distribution centers on the strand / angles when the peptide is embedded in the hg / c region of the membrane ( lower left panel of figure s2 ) . such a conformational change may have some effect on the free energy profile of the peptide as it travels through the membrane . yet , it is difficult to quantify this effect because we have not imposed any constraints on the / angles of the peptide . in the upper leaflet , the nitrogen heteroatom points toward the interfacial regions while the peptide moves through the membrane ; while in the lower leaflet , the ring nitrogen consistently points toward the core of the membrane ( see figure 2 and figure 3 ) . this preferential orientation results in distinct and deeper energy minima at the interfacial regions of the upper leaflet while the corresponding minima in the lower leaflet are shallow and less distinct ( see figure 1 and figure 2b ) . the location of the minima and the orientation of the indole ring in the upper leaflet indicate that the ring is in a prime position to interact with the glycerol carbonyls and other hydrogen bonding elements of the lipid head groups . the importance and extent of hydrogen bonding between the indole ring and the membrane interfacial region has been addressed in a previous study.(26 ) here , the nitrogen heteroatom forms a significant number of hydrogen bonds with the membrane , and it does so to a greater extent where the minima occur along the reaction coordinate ( see figure 4 ) . in the upper leaflet , where the minima are deeper and more distinct , a larger number of hydrogen bonds are formed as compared with the minima in the lower leaflet . ( a ) density distributions of the tryptophan indole nitrogen in the upper and lower leaflets of four simulation windows . dashed lines indicate where the peptide was constrained ( via the indole ring ) in that particular simulation . ( b ) the asymmetric free energy profile in the membrane ( dashed line ) and the same profile shifted to reflect the nitrogen heteroatom position ( dark heavy line ) . at each point , the nitrogen is distributed to the right ( more positive z ) of the indole center ; but the magnitude of that shift is not equal in all parts of the profile . snapshots of wl5 in the upper ( above ) and lower ( below ) leaflets of the membrane . the membrane is shown as a transparent surface with the lipid head groups colored pink and the tails brown . the peptide is shown as van der waals spheres with the indole ring highlighted in yellow . the insets show a close - up of the indole ring orientation , with carbon , hydrogen , and nitrogen atoms colored in cyan , white , and blue , respectively . average number of hydrogen bonds formed between the indole nitrogen and the hydrogen bonding elements of the lipids , as a function of the peptide s position along the reaction coordinate . the cutoff criterion was set to elements within 3 and hydrogen bonding angles of 40. analysis with respect to orientation of the entire peptide reveals a tilt in the peptide axis ( see figure 5 ) , an observation common in helical transmembranepeptide systems.(27 ) as it adheres and inserts into the membrane , the peptide axis stabilizes at an angle near 70 , which is off parallel to the membrane plane ( an angle of 90 ) . such a tilt allows the leucine residues to interact with the hydrophobic part of the membrane while maintaining the indole ring position in the interfacial region . this appears to be the general strategy of transmembrane peptides , in that they are designed to exploit both the hydrophilic and the hydrophobic parts of the membrane during the insertion process . the orientations of the whole peptide and the tryptophan side chain argue that the pmf in the upper leaflet represents a more plausible path of peptide insertion . but in reality , the peptide could approach the membrane in a variety of orientations . also , it may be the case that the peptide binds the membrane surface in a random initial orientation and then rearranges prior to further insertion and stabilization . peptide tilt as a function of position along the z axis , with the membrane centered on 0 . inset shows how the angle is defined by the vector fitted to contain the alpha carbons of the peptide ( light arrow ) and the vector representing the membrane normal ( dark arrow ) . however , we do not have any a priori knowledge of the peptide orientation or information on its conformational dynamics at the solventmembrane interface . furthermore , the constraint applied at the trp residue during the umbrella sampling does not allow for a spontaneous reorganization . it is therefore impossible to determine the relative weights of the two profiles , and here we assume that the lower- and upper - leaflet orientations are equally probable . in other words , because the molecular dynamics simulations in each leaflet are separate and independent , one can consider the data from the upper and lower leaflets as twice the sampling of the same system . thus , we symmetrize the free energy profile ( heavy blue line in figure 1 ) and allow this result to reflect the sampling of the entire system ( see materials and methods ) . this symmetric profile is used for all further calculations and discussion , but we note that this symmetry is due purely to the homogeneity of the model membrane used in the simulations . in biological heterogeneous membranes , the peptide begins its approach toward the membrane from a region of bulk solvent , where the pmf plateaus ( 35 ) . this plateau indicates that the peptide does not interact with the membrane and instead experiences a purely aqueous environment . the average pmf in this region ( aqua - colored in figure 1 ) is 10.1 kcal mol . this value will be used as the reference point from which the change in free energy ( g ) will be calculated . in other words , as the peptide travels closer to the membrane , at about 30 from the center , the free energy rapidly declines as the peptide encounters the solventheadgroup ( s / hg ) interface . such a drop in the free energy is often encountered when a hydrophobic solute encounters a like environment.(28 ) inside this s / hg region , the free energy profile continues to decline until it reaches a shallow minimum near 17 , and the barrier to escape this minimum is approximately 0.5 kcal mol . [ this barrier seems insignificant given the sampling error of this method ( see materials and methods ) , and it should be greater . nonetheless , we know from previous experiment and simulation that a minimum in the free energy profile occurs here . ] the computed free energy change as the peptide inserts into the s / hg interface is gs / hg = 8.3 kcal mol . thus , the peptide has a tendency to localize in the s / hg region , and this can likely be attributed to the partial hydrophilic nature of the trp residue . the peptide descends further into the headgroupcore ( hg / c ) interface and encounters a global minimum at about 7 and gains a further 2 kcal mol in free energy . the total free energy change from the bulk solvent to the hg / c region ( ghg / c ) is 10.2 kcal mol . a local maximum occurs in the core of the membrane ( 0 ) , and the energy barrier as measured from the hg / c region is approximately 1.0 kcal mol . the root - mean - square fluctuation ( rmsf ) demonstrates how much the peptide deviates from its average conformation and is plotted in figure 6a as a function of the peptide location along the membrane normal . one can conclude that the peptide is more flexible and samples a wider region of conformational space ( has a larger rmsf ) as it travels deeper into the membrane core . this flexibility is also demonstrated by the root - mean - square deviation ( rmsd ) of the peptide structure , as seen in figure s3 of supporting information . an alternative view of the rmsf in figure 6b further shows that the side chains of the peptide are predominantly responsible for this flexibility . in particular , the trp side chain has the largest rmsf when deep in the membrane core . to the extent that peptide flexibility can be correlated with entropy,(29 ) one can infer that the peptide exhibits increased entropy as it inserts into the membrane . this results in a more negative ( or favorable ) contribution to the free energy change and explains why the energy barrier in the core is not greater in the calculated profile . therefore , a small barrier seems to suggest that the peptide can occasionally translate across the membrane center and shift positions between the two leaflets . yet , as wimley and white have demonstrated and as we shall explore further below , individual units of this model peptide aggregate in the membrane and form a supramolecular structure , a process that can counteract the tendency of a lone peptide to transfer from leaflet to leaflet . root mean square fluctuation ( rmsf ) of the peptide by atom number and as a function of position along the z axis of the simulation box . atom numbers 13 correspond to the acetyl group on the n - terminus ; 424 correspond to the tryptophan residue ; 2569 correspond to the leucine residues ; and 7072 correspond to the amide group on the c - terminus . ( a ) surface representation , where the red halo shows the maximum rmsf achieved by the indole ring near the core of the membrane . the free energy profile presented here is that of a single peptide traveling through the membrane and is thus not reflective of the entire biological process . if the final state of wl5 in the membrane is not monomeric but rather an aggregate form , then it behooves us to study this aggregation process and the pathways that lead to it . although we have not simulated the actual aggregation , the presented work , coupled with previous simulations and experimental evidence , can shed some light on the mechanism and potential pathways of aggregation . figure 7 shows a proposed thermodynamic cycle for the insertion and assembly of wl5 in the membrane . in this model , the initial state of the peptide in an aqueous environment is monomeric , and the final state in membrane is an assembly of several monomers ( top left and bottom right of figure 7 , respectively ) . the difference in the free energy between these two states as obtained by wimley and white is gexp = 5.3 kcal mol ( exp = experimental).(13 ) [ this is assuming that the transfer from monomeric aqueous to membrane aggregate form is an equilibrium process , and that the experimental result is a reflection of this . ] as suggested by grossfield et al . , the computed free energy can be coupled to this experimental value via a correction term that accounts for the difference in the peptide and lipid concentrations between simulation and experiment.(30 ) proposed thermodynamic cycle of peptide insertion and supramolecular assembly inside a model membrane . the yellow arrows depict a pathway in which single peptides insert first into the deep headgroupcore ( hg / c ) interface and then aggregate to form the final structure . the red arrows mark a less likely ( yet plausible ) path , in which the peptide aggregates first in solution , and then the aggregate as a whole inserts into the membrane . the green arrows qualitatively describe an intermediary pathway , in which individual peptides insert first into the aqueous or solventheadgroup ( s / hg ) interface , aggregate to some extent , and then insert deeper into the membrane . each of these steps can be described by a free energy change ( g , aggr = aggregation ) . following the notation of grossfield , gr and gmix are the correction terms for the peptide and lipid ( respectively ) concentration differences between simulation ( sim ) and experiment ( exp ) . is the mole fraction of the species in question ( either peptide or lipids ) . this entire correction term , gcorr , is calculated to be + 3.0 kcal mol ( see materials and methods for further information ) and must be added to the computational results . in terms of insertion and aggregation , figure 7 shows two paths on opposite ends of the spectrum . in the yellow path , each peptide inserts individually into the deeper membrane interface . the aggregation step then follows : equation 5 is the corrected version of eq 4 , where the correction term has been added to computational result ghg / c = 10.2 kcal mol . if we can set gyellowpath to be the experimental value determined by wimley and white ( 5.3 kcal mol ) , the aggregation term is then calculated to be + 1.9 kcal mol . the red path in figure 7 illustrates the opposite approach , in which individual peptides in aqueous solution first assemble , and then the whole aggregate inserts into the membrane . the first leg of this pathway is plausible and has a negative free energy change , because hydrophobic peptides in an aqueous environment are energetically more stable in aggregate instead of monomeric form.(31 ) yet , the stability and energetic gain of the aggregate in solution could deter the adhesion and insertion of the aggregate into the membrane , thus making the second leg of the red path less likely . furthermore , if the orientation of each monomeric peptide can affect the thermodynamics of the insertion process ( as discussed above ) , it may become energetically costly for the peptide units of the complex to reorganize and assume the correct orientation . others have also shown that transmembrane proteins do not fold entirely or form complex structures before inserting into a membrane.(32 ) instead , it is more likely that a transmembrane peptide adsorbs on the membrane surface , aggregates to some extent , and then inserts into the membrane . as the insertion event proceeds , it is likely that the membrane continues to shape the peptide aggregate structure until it achieves a final stable form in the membrane host . note that membranes exhibit the mechanical properties to do just this.(33 ) for our model system , this is depicted in the green path , in which the monomeric peptide first adheres to the s / hg interface , aggregates , and then inserts : equation 7 represents the corrected version , with the correction term added again to the computational result , gs / hg = 8.3 kcal mol . the last two terms on the left - hand side of equation 7 are the unknowns , and we can not calculate the exact contribution from gaggr(s / hg ) or gaggr(insert ) . again , if we equate ggreenpath to the experimental value of 5.3 kcal mol , the sum of these unknown terms can be calculated , gaggr(s / hg ) + gaggr(insert ) = 0 kcal mol . thus , in the yellow path , the aggregation free energy change is positive and indicative of an unfavorable process . in other words , if the monomeric form of the peptide were to insert deep into the hg / c region of the membrane , aggregation would not be likely . in the green path however , the collective aggregation term is 0 , indicating an equilibrium ( and more likely ) process . we have shown the following relation : where the braces group the unknown terms of each path , as described above . the results suggest that the green path in figure 7 , where the peptide first adheres to the solventheadgroup interface , aggregates , and then inserts , more closely represents the in vitro mechanism of insertion and organization of wl5 . furthermore , following the previous discussion of the red path in which we stated that a formed aggregate would have difficulty inserting into the membrane , we can speculate that gaggr(insert ) > 0 and thus gaggr(s / hg ) < 0 . in other words , aggregation occurs immediately upon peptide binding to the membrane , in the solventheadgroup region . the structure of the solvated peptidemembrane system was obtained from a previous study , in which we explored the properties of the system in a 75 ns md simulation.(25 ) snapshots at the end of this simulation , at which point , the peptides were embedded within the headgroup region of the membrane , were used as starting configurations to initiate 50 different all - atom simulations each representing a window in an umbrella sampling scheme . two different snapshots of the peptide were employed ( one for each leaflet of the membrane ) ; in other words , the 25 simulation windows within a leaflet were initiated with the same initial peptide conformation . these two snapshots are taken to be the equilibrated conformation of the peptide when embedded in the solventheadgroup interfacial region of the membrane . in total then , the 50 simulation windows consisted of a wl5 peptide ( acetylated and amidated at its n- and c - termini , respectively ) , a preconstructed bilayer of 128 dimyristoylphosphatidylcholine ( dmpc ) lipids,(20 ) and 6631 water molecules , resulting in a 25 000 atom system assembled in a simulation box of 60 60 95 . the membrane was positioned at the center of the box , leaving approximately 23 for the solvent on either side . each simulation corresponds to a window of width 1.9 such that the position of wl5 was different in each simulation . in the first window , wl5 was constrained ( along the z axis ) in the bulk solvent at the upper half of the box and well away from the membrane surface . in the next , wl5 was moved by 1.9 closer to the membrane and constrained at that position . in subsequent simulations , wl5 was moved closer to the center of the simulation box each time stepped along the z axis by the increment of 1.9 . the constrained molecule thus spans the entire reaction coordinate ( in the z direction ) of approximately 95 . for organizational purposes , the simulation box is divided into two halves along the z axis , representing the upper ( positive z ) and lower ( negative z ) leaflets with respect to the membrane center at z = 0 . among the 25 simulation windows in each leaflet , 12 had the peptide constrained in solvent and 13 were carried out with the peptide constrained at various z locations in the membrane . the initial configuration of the peptide differs between the two leaflets but is the same among the 25 simulation windows of each leaflet . the reaction coordinate was defined by the separation along the z axis between the centers - of - mass of the indole chromophore , the tryptophan side chain , and the membrane . wl5 was constrained by a harmonic potential of force constant 500 kj mol nm ( 1.20 kcal mol ) . first , unfavorable contacts were relieved by two cycles of 5000 steps steepest descent followed by 5000 steps of conjugate - gradient energy minimizations , each with the peptide held fixed and set free . second , except when the peptide is in bulk solvent , an annealing step was conducted to equilibrate the lipid tails around the peptide . with the peptide and lipid head groups restrained , the system was gradually heated ( over 1 ns ) from 310 k ( the temperature of the initial snapshot ) to 410 k , then gradually cooled back down to 310 k. third , adding pressure coupling , a restrained md was performed on all 50 windows for 1 ns , with the restraint applied to nonsolvent molecules . fourth , with only the indole ring of tryptophan constrained to its selected position , production runs commenced . those with the peptide located outside of the membrane were sampled for 5 ns , while those with the peptide somewhere in the membrane were sampled for 25 ns . in total , including all pre- and post production runs , approximately 850 ns of all - atom md simulations have been carried out . a time step of 2 fs was used while coordinates / velocities were recorded every 500 steps ( 1 ps ) full electrostatics were calculated using the particle mesh ewald ( pme ) method , with coulombic and van der waals cut - offs of 0.9 and 1.4 nm , respectively . a sixth - order spline interpolation was used for pme along with a tolerance of 1 10 . nearest neighbor lists were updated every 10 steps using the grid method and periodic boundary conditions ( in xyz ) were employed with a cutoff of 0.9 nm . berendsen temperature coupling was used , with a reference temperature of 310 k and a coupling of 0.1 ps . a semi - isotropic pressure coupling scheme , in which the xy dimensions are coupled together while the z direction is allowed to fluctuate independently , was used with a reference pressure of 1.0 bar , a pressure coupling of 0.5 ps , and a compressibility of 4.5 10 bar . the simulations were performed with the gromacs md package.(34 ) the ffgmx force field was used for the peptide and the spc water model was used as the solvent . the lipid parameters of berger et al . were employed for the dmpc lipids.(35 ) the analysis of the simulation trajectories was completed using the various gromacs tools . for further details regarding these tools and specific procedures in running gromacs md simulations ; see the gromacs manual at http://www.gromacs.org/. visualization and rendering were done by the visual molecular dynamics ( vmd ) application.(36 ) given the construction of the simulation windows , one might expect the resulting profile in figure 1 to be perfectly symmetric about the membrane center . in other words , the profile in one leaflet should be a mirror image of the other provided that the peptide exhibits the same orientation while penetrating through both leaflets of a homogeneous membrane . as mentioned in the text , the free energy profile of figure 1 is not exactly symmetric , and this was attributed mainly to the starting conformation of the peptide in the simulation windows of each leaflet . yet , even if the peptide conformation was the same in each leaflet , it is doubtful that one could achieve great symmetry , simply due to convergence limitations inherent in md simulations . in fact , the asymmetric profile may be used as a convergence criterion with respect to certain free energy calculations . for instance , the values of the computed ghg / c in the upper and lower leaflets are within 1 kcal mol of each other while the values of gs / hg differ more significantly . this could signify that the simulation of deep insertion ( from bulk solvent into the deep interface ) produces a more converged result than insertion into the solventheadgroup ( s / hg ) interface . one could reason that this is the case because the s / hg interface is more chemically diverse and dynamic , thus requiring more sampling time for energetic calculations . in figure 1 , about the center of the profile ( at 0 ) , the average was taken between the two points on either side of the profile . for instance , if the free energy was 1.8 kcal mol at + 10 ( upper leaflet ) and 1.5 kcal mol at 10 ( lower leaflet ) , the free energy in the symmetric plot at 10 is taken to be 1.65 kcal mol . the asymmetric free energy profile in figure 1 the differences at mirror sites along the reaction coordinate ( for instance , at 7 ) reveal a sampling error on the order of 0.51.0 kcal mol . as reported by wimley and white , the experimental concentration of the wl5 peptide is ( at most ) 100 m.(13 ) using this concentration , and that of water to be 55.5 m ( or 55.5 10 m ) , we can calculate the mole fraction of the peptide in the experiment to be exp , peptide = 1.80 10 . in each simulation , we have one peptide , in either the upper or lower leaflet of the system . because of periodic boundary conditions , the peptide is solvated in the total solvent of 6631 water molecules . thus , the mole fraction of the peptide in the simulation is sim , peptide = 1.51 10 . substituting these mole fractions along with the simulation temperature ( 310 k ) into the expression for gr , we arrive at + 2.73 kcal mol , which corrects for the differences in peptide concentration between simulation and experiment . to calculate the mole fractions of the lipids , a volumetric ratio is computed in a fashion similar to grossfield et al . taking the average thickness of a dmpc membrane to be 36 and the cross - sectional area to be 60 60 ( per simulation setup ) , we compute the volume occupied by the membrane to be 1.30 10(5 ) . when the ratio of the membrane volume to the box volume is computed , we obtain sim , lipids = 0.38 ; we also use exp , lipids = 0.24 , for an experimental vesicle concentration of 4 mm.(29 ) these mole fractions result in gmix = + 0.28 kcal mol , and this corrects for the differences in lipid concentration between simulation and experiment . the combined correction term is then the sum , gr + gmix = gcorr + 3.0 kcal mol .

a variety of biomolecules mediate physiological processes by inserting and reorganizing in cell membranes , and the thermodynamic forces responsible for their partitioning are of great interest . recent experiments provided valuable data on the free energy changes associated with the transfer of individual amino acids from water to membrane . however , a complete picture of the pathways and the associated changes in energy of peptide insertion into a membrane remains elusive . to this end , computational techniques supplement the experimental data with atomic - level details and shed light on the energetics of insertion . here , we employed the technique of umbrella sampling in a total 850 ns of all - atom molecular dynamics simulations to study the free energy profile and the pathway of insertion of a model hexapeptide consisting of a tryptophan and five leucines ( wl5 ) . the computed free energy profile of the peptide as it travels from bulk solvent through the membrane core exhibits two minima : a local minimum at the watermembrane interface or the headgroup region and a global minimum at the hydrophobichydrophilic interface close to the lipid glycerol region . a rather small barrier of roughly 1 kcal mol1 exists at the membrane core , which is explained by the enhanced flexibility of the peptide when deeply inserted . combining our results with those in the literature , we present a thermodynamic model for peptide insertion and aggregation which involves peptide aggregation upon contact with the membrane at the solventlipid headgroup interface .

Introduction Results and Discussion Materials and Methods

PMC4836393

probes capable of recognizing specific mixed - sequence double - stranded dna ( dsdna ) regions have been long - sought - after as they can be developed into tools that enable modulation of gene expression at the transcriptional level , gene editing , and detection of specific genetic signatures . early examples of dsdna - targeting probes include triplex - forming oligonucleotides ( tfos ) and peptide nucleic acids ( pnas ) , as well as minor groove binding polyamides . unfortunately , only a subset of the possible target regions is available for recognition by these probes due to requirements for exclusive purine content ( tfos / pnas ) or short target regions ( polyamides ) . consequentially , significant efforts have been devoted to develop alternative approaches , which has resulted in tfos and pnas with reduced target site limitations . more recently , engineered proteins such as zinc finger nucleases , transcription activator - like effector nucleases ( talens ) and in particular crispr / cas9 systems , have gained a tremendous amount of attention , despite mounting concerns regarding recognition specificity and cellular delivery . another class of compounds that has emerged from these efforts are the so - called pseudocomplementary dna and pna ( pcdna / pcpna ) , in which a short dna or pna duplex is modified to contain pseudocomplementary base pairs between 2-thiothymine and 2,6-diaminopurine ( figure 1a ) . the steric clash between the 2-thio and the 2-amino group perturbs and weakens the hydrogen bonding between these moieties , resulting in a destabilized probe duplex . in contrast , 2-thiothymine and 2,6-diaminopurine form stable base pairs with canonical adenine and thymine , respectively . the difference in thermodynamic stability between probe duplexes and duplexes between individual probe strands and complementary dna ( cdna ) allows for double - duplex invasion of dsdna target regions under certain conditions ( figure 1b ) . thus , pcdna can recognize terminal target regions , while pcpna also recognize internal target regions of dna duplexes , albeit at low ionic strengths . however , a recent study has suggested that recognition of mixed - sequence dsdna regions by pcpna may be possible under the highly viscous conditions found in the nucleus . we , and later others , have pursued an alternative strategy for the construction of energetically activated double - stranded probes for recognition of mixed - sequence dsdna regions , which is based on forced intercalation of aromatic moieties . our invader probes are short dna duplexes containing + 1 interstrand zipper arrangements of intercalator - functionalized nucleotides ( figure 1c ; for a definition of the zipper nomenclature , see experimental section ) . this motif , which we denote as an energetic hotspot , forces two intercalators into the same region of the duplex , resulting in unwinding and destabilization as the nearest neighbor exclusion principle is violated . according to this principle , the space between the two nearest base pairs on either side of a bound intercalator , will not be bound by a second intercalator due to limitations in local helix expandability ( every intercalation event unwinds the duplex by 3.4 ) , and/or to avoid disruption of highly stable stacking interactions between the first bound intercalator and neighboring nucleobases . on the other hand , each of the two strands comprising an invader probe display very high affinity toward cdna since the intercalators stack strongly with the neighboring base pairs , acting as molecular glue ( figure 1c ) . we have previously demonstrated that the differences in thermostability between invader probes and probe - cdna duplexes can drive mixed - sequence recognition of isosequential dna duplexes , dna hairpins , and chromosomal dna targets . initially , 2-n-(pyren-1-yl)-2-amino--l - lna ( locked nucleic acid ) monomers were used to construct the energetic hotspots of invader probes . however , the challenging synthesis of these building blocks , prompted us to conduct a search for more feasible structural and functional mimics , which , among others , resulted in the identification of 2-n-(pyren-1-yl)methyl-2-n - methyl-2-aminouridine monomer x as a next - generation invader modification ( figure 1a ) . access to building blocks that are based on simpler sugar scaffolds has facilitated extensive structure property relationship studies aiming at improving the recognition efficiency of invader probes . in the present study , we set out to study if the dsdna - recognition potential of invader probes can be increased further through incorporation of pseudocomplementary base pairs . we hypothesized that these chimeric pseudocomplementary invader probes will be more energetically labile , yet form even more stable duplexes with cdna than either canonical invader or pcdna probes , leading to more favorable energetics for dsdna - recognition ( figure 1b ) . toward this end , two different approaches were followed . in the first , we wanted to integrate the intercalator as part of a pseudocomplementary nucleotide , which led to the identification of 2-n-(pyren-1-yl)methyl-2-n - methyl-2-amino-2-thiouridine monomer y as a target ( figure 1a ) . incorporation of this monomer in a + 1 interstrand zipper arrangement opposite of conventional 2,6-diaminopurine dna d monomers would furnish a double - stranded probe with a pseudocomplementary energetic hotspot ( dy probes ) ( figure 1b ) . in the second approach , we separated the two key structural features by using conventional 2-thiothymine dna monomer s and 2,6-diaminopurine dna monomer d , alongside energetic hotspots comprised of the conventional invader monomer x ( dsx probes ) ( figure 1b ) . we demonstrate that the latter type of probes is particularly interesting for dsdna - recognition applications . ( b ) representation of energy levels of different dsdna - targeting probes and the corresponding duplexes with cdna ( only one probe - target duplex is shown ) . note , the large difference in energy between pc - invader : cdna duplexes and pc - invader probe duplexes . the key 2-n-(pyren-1-yl)methyl-2-n - methyl-2-amino-2-thiouridine phosphoramidite 6 was obtained from known nucleoside 1 , following a similar general strategy as was used for the synthesis of 2-o-[2-(methoxy)ethyl]-2-thiothymidine ( scheme 1 ) . nucleoside 1obtained in 61% yield over six steps from uridine was first subjected to a sequence of protecting group manipulations , i.e. , 3-o - acetylation , 5-o - detritylation and 5-o - methanesulfonylation , to afford nucleoside 2 in 55% yield over three steps . prolonged refluxing in anhydrous ethanol in the presence of sodium bicarbonate , results in the formation of 2-o - ethyluridine derivative 3 in 52% yield , presumably via a nucleophilic opening of an o2,o5-anhydrouridine intermediate . subsequent o5-dimethoxytritylation of 3 using standard conditions affords nucleoside 4 in 88% yield , which upon treatment with h2s - saturated pyridine in the presence of 1,1,3,3-tetramethylguanidine provides 2-thiouridine derivative 5 in 82% yield . treatment of nucleoside 5 with 2-cyanoethyl - n , n - diisopropylchlorophosphoramidite and n , n - diisopropylethylamine affords target phosphoramidite 6 in 81% yield , corresponding to an overall yield of 17% from nucleoside 1 . dmtr = 4,4-dimethoxytrityl ; py = pyren-1-yl ; ms = methanesulfonyl ; tmg = 1,1,3,3-tetramethylguanidine ; pcl reagent = phosphoramidite 6 was used to incorporate monomer y into oligodeoxyribonucleotides ( ons ) via automated solid - phase dna synthesis . extended hand - coupling ( 15 min ) and the use of 4,5-dicyanoimidazole as an activator resulted in stepwise coupling yields of 95% . ons modified with monomer x were synthesized as previously described ( 15 min coupling , 5-[3,5-bis(trifluoromethyl)phenyl]-1h - tetrazole as an activator , 99% coupling yield ) . the corresponding phosphoramidites of monomers s and d were obtained from commercial sources and incorporated into ons using the conditions for incorporation of monomer y ( stepwise coupling yields > 95% ) . to prevent desulfurization in ons modified with y or s monomers , nucleotide phosphite to phosphate oxidation was performed using tert - butylhydroperoxide / ch3cn / h2o ( 10 min ) rather than the standard aqueous iodine solution . the identity and purity of the modified ons were established through maldi - tof ( table s1 ) and ion - pair reverse - phase hplc ( > 90% purity unless otherwise noted ) , respectively . pyrene - functionalized 2-thiouracil monomer y was incorporated into the same 9-mer mixed - sequence ons that was previously used for evaluation of invader monomer x. thermal denaturation temperatures ( tm s ) of duplexes between y - modified ons and cdna or crna were determined in a medium salt phosphate buffer ( [ na ] = 110 mm ) and compared relative to unmodified and corresponding x - modified duplexes . the resulting denaturation curves display the expected monophasic sigmoidal transitions ( figure s1 ) . duplexes between y1y4 and cdna are significantly more stable than unmodified reference duplexes ( tm between + 2.5 and + 11.5 c , table 1 ) , whereas heteroduplexes with crna are much less stable ( tm between 6.5 to + 2.5 c , table s2 ) . ons in which y monomers are flanked by 3-purines result in greater duplex stabilization than ons with 3-flanking pyrimidines ( e.g. , compare tm s for y1 and y3 , table 1 ) . this sequence dependence , along with the prominent dna selectivity ( table s3 ) , is typical for ons modified with intercalating pyrene moieties . surprisingly , y - modified ons form slightly less stable duplexes with cdna than their x - modified counterparts ( compare tm s of x1x4 and y1y4 , table 1 ) , which suggests that the binding modes responsible for the stabilizing contributions of the pyrene and 2-thiouracil moieties are not fully compatible . to generate invader probes with pseudocomplementary energetic hotspots , we synthesized ons in which 2-amino-2-deoxyadenosine monomers flank monomer y ( i.e. , the dy series ) . replacing regular 2-deoxyadenosines with d monomers increases the cdna / crna affinity of y - modified ons by 15 c , presumably due to stabilization from the extra hydrogen bond in d : t base pairs relative to normal a : t pairs ( e.g. , compare tm for y1 and dy1 , table 1 and table s2 ) . similar relative increases in cdna affinity are observed when 2-deoxyadenosines are replaced with d monomers in otherwise unmodified ons ( see tm for d1d4 vs cdna , table 1 ) , which is in agreement with previous studies . tm = change in tm relative to reference duplex dna1:dna2 ( tm 29.5 c ) , where dna1 : 5-gtg ata tgc and dna2 : 3-cac tat acg . tm s are determined as the maximum of the first derivative of melting curves ( a260 vs t ) recorded in medium salt phosphate buffer ( [ na ] = 110 mm , [ cl ] = 100 mm , ph 7.0 ( nah2po4/na2hpo4 ) ) , using 1.0 m of each strand . reported tm s are averages of at least two measurements within 1.0 c ; a = adenin-9-yl dna monomer , c = cytosin-1-yl dna monomer , g = guanin-9-yl dna monomer , and t = thymin-1-yl dna monomer . for structures of monomers next , we set out to study dna duplexes with different interstrand zipper arrangements of y monomers and dy segments to identify probe architectures that are strongly activated for dsdna - recognition . as expected from our previous studies on invader probes , y1:y3 , which features a + 1 interstrand zipper of y monomers , is much more thermolabile than y1:y2 featuring a 1 interstrand zipper ( table 2 ) , or duplexes between y - modified ons and cdna ( table 1 ) . the destabilization is likely a consequence of the nearest neighbor exclusion principle being violated . in line with our hypothesis , introduction of a 2,6-diaminopurine d monomer opposite of the pyrene - functionalized 2-thiouracil y monomer , decreases the tm s of the duplexes ( e.g. , compare tm of dy2:dy3 and y1:y2 , table 2 ) . interestingly , the destabilizing effect of the pseudocomplementary base pairs is more pronounced when the y monomers are positioned in a 1 zipper orientation ( drop in tm of 9.5 c from y1:y2 to dy2:dy3 , relative to drop of 2.0 c from y1:y3 to dy1:dy4 , table 2 ) . this indicates that the pseudocomplementary energetic hotspot architecture of dy1:dy4 does not fully harness the activating effects from both structural elements . the tm - based conclusions were largely corroborated by the gibbs free energies associated with duplex formation , which were derived from denaturation curves via line fitting ( table 2 ) . thus : ( i ) duplexes between y - modified ons and cdna are much more stable than unmodified reference duplexes ( g between 16 and 7 kj / mol , first and second g columns , table 2 ) due to more favorable enthalpy ( h between 76 and 44 kj / mol , table s7 ) . ( ii ) in comparison , the corresponding x - modified duplexes are slightly more stable , while the d - modified duplexes are far less stable ( e.g. , compare g for y1:cdna , x1:cdna and d1:cdna , table 2 ) . ( iii ) duplexes between ons with dy motifs and cdna are generally less stable than the corresponding y - modified duplexes ( e.g. , compare g for y1:cdna and dy2:cdna , table 2 ) . this contrasts the tm - based conclusions ( table 1 ) , but likely reflects the different entropies of these duplexes ( table s8 ) , leading to different temperature dependencies of the gibbs free energies . ( iv ) formation of y1:y3 ( + 1 zipper ) is less thermodynamically favorable than y1:y2 ( 1 zipper ) ( third g column , table 2 ) . ( v ) duplexes with pseudocomplementary energetic hotspots are slightly less stable than the corresponding duplexes featuring only regular energetic hotspots ( e.g. , compare g for dy1:dy4 and y1:y3 , table 2 ) . the energetic nature of dy1:dy4 is the result of particularly unfavorable enthalpy ( h = + 95 kj / mol , table s7 ) , likely reflecting a violation of the nearest neighbor exclusion principle and concomitant formation of destabilizing pseudocomplementary base pairs . ( vi ) pc - dna without any energetic hotspots range between being slightly more stable to slightly less stable than unmodified duplexes ( e.g. , see g for d1:y2 and y1:d3 , table 2 ) . consequentially , + 1 zipper duplex y1:y3 is much more energetically activated than 1 zipper duplex y1:y2 , as gauged by the free energy available for recognition of isosequential dsdna targets grec293 ( ona : onb ) = g ( ona : cdna ) + g ( cdna : onb ) g ( ona : onb ) g ( dsdna ) , where ona : onb denotes a double - stranded probe and dsdna is the isosequential dsdna target for ona : onb ( compare grec293 for y1:y2 and y1:y3 , table 2 ) . dy1:dy4 , featuring a pseudocomplementary energetic hotspot , is slightly more energetically activated for dsdna - recognition than y1:y3 ( compare grec293 for dy1:dy4 and y1:y3 , table 2 ) . as expected , dy2:dy3 , which also features two pseudocomplementary base pairs but has an 1 interstrand zipper arrangement of y monomers , is far less activated for dsdna - recognition ( compare grec293 for dy2:dy3 and dy1:dy4 , table 2 ) . pc - dna without energetic hotspots are also far less activated for dsdna - recognition ( e.g. , compare grec293 of y1:d3 and dy1:dy4 , table 2 ) . unexpectedly , dy1:dy4 has lower thermodynamic dsdna - recognition potential than conventional invader probe x1:x3 ( compare grec293 for dy1:dy4 and x1:x3 , table 2 ) , which likely is due to two factors : ( i ) intercalation of the pyrene moieties perturbs the local duplex geometry , which weakens the normally stabilizing base pairs between 2,6-diaminopurine : thymine ( d : t ) and 2-thiouracil : adenine ( y : a ) , leading to lower - than - expected cdna - affinity of dy - modified ons ( e.g. , compare g for x1:cdna , y1:cdna and dy1:cdna , table 2 ) , and ( ii ) + 1 interstrand zipper arrangements of nucleotide monomers with intercalating pyrene moieties perturb local duplex geometries , which , in turn , are likely to reduce the steric clash between 2,6-diaminopurine and 2-thiouracil normally occurring in pseudocomplementary base pairs , resulting in less pronounced probe destabilization . it is also possible that the pseudocomplementary base pairs of dy1:dy4 interfere with the forced and destabilizing intercalation of the two pyrene moieties ; this is supported by the uv vis absorption characteristics of dy1:dy4 . normally , dna duplexes with + 1 interstrand zipper motifs of intercalating pyrene - functionalized monomers exhibit markedly blue - shifted pyrene absorption relative to dna duplexes with other interstrand zipper motifs , which is indicative of reduced pyrene - nucleobase interactions due to a locally perturbed duplex geometry ( e.g. , compare max for x1:x3 relative to x1:x2 , table 2 ) . the same trend is observed for y1:y3 ( compare max for y1:y3 relative to y1:y2 ) , but the trend is less pronounced for dy1:dy4 ( compare max for dy1:dy4 relative to dy2:dy3 ) , indicating that dy1:dy4 is not strongly perturbed . zp = zipper . for conditions of thermal denaturation , see table 1 . grec293 ( ona : onb ) = g ( ona : cdna ) + g ( cdna : onb ) g ( ona : onb ) g ( dsdna ) . absorption spectra were recorded at t = 10 c using each strand at 1.0 m concentration in tm buffer . data for x1:x2 and x1:x3 are from ref ( 32 ) and are included to facilitate comparison . on the basis of the observed grec293 values , we decided to determine the dsdna - recognition properties of y1:y3 and dy1:dy4 relative to benchmark invader x1:x3 using an electrophoretic mobility shift assay from our earlier studies . thus , a digoxigenin ( dig ) labeled dna hairpin ( dh)comprised of a 9-mer double - stranded mixed - sequence stem , which is linked by a t10 loop was used as a model dsdna target ( figure 2a and 2b ) . room temperature incubation of dh1 with y1:y3 , dy1:dy4 , or x1:x3 in a hepes buffer of considerable ionic strength , results in dose - dependent formation of a ternary recognition complex as evidenced by the emergence of a slower migrating band on nondenaturing page gels ( figure 2c ) . response curves reveal that x1:x3 , y1:y3 , and dy1:dy4 have c50 values of 0.8 m , 2.8 m , and 1.5 m , respectively ( figure 2d ) , which mirrors the trend in grec293 values ( table 2 ) . the binding specificities of y1:y3 , dy1:dy4 , and x1:x3 were examined by incubating a 200-fold molar excess of the probes with dna hairpins dh2dh7 , which deviate in the nucleotide sequence at one position relative to the invader probes ( figure 2b ) . no recognition was observed , demonstrating that invader - mediated dsdna - recognition proceeds with excellent binding specificity ( figure 2e ) . ( a ) illustration of recognition process ; ( b ) sequences and thermal denaturation temperatures of dna hairpins with isosequential ( dh1 ) or nonisosequential stems ( dh2dh7 ) ; underlined nucleotides indicate sequence deviations relative to probes ; ( c ) representative electrophoretograms of recognition of dh1 using 1- to 500-fold molar excess of y1:y3 or dy1:dy4 ; ( d ) dose response curves ( average of at least three independent experiments ; error bars represent standard deviation ) ; ( e ) electrophoretograms illustrating incubation of dh1dh7 with 200-fold molar excess of x1:x3 , y1:y3 , or dy1:dy4 . experimental conditions for electrophoretic mobility shift assay : separately preannealed targets ( 34.4 nm ) and probes ( variable concentrations ) were incubated for 1216 h at ambient temperature in 1x hepes buffer ( 50 mm hepes , 100 mm nacl , 5 mm mgcl2 , 10% sucrose , 1.4 mm spermine tetrahydrochloride , ph 7.2 ) and then resolved on 16% nondenaturing page ( 70 v , 2.5 h , 4 c ) using 0.5 tbe as a running buffer ( 45 mm tris , 45 mm boric acid , 1 mm edta ) ; dig : digoxigenin . the above results suggest that incorporation of pseudocomplementary energetic hotspots i.e . , 5-yd-3:3-dy-5 cassettes provide less of a benefit for dsdna - recognition than originally anticipated . at this stage , we hypothesized that the two structural elements that activate invader and pc - dna probes for dsdna - recognition the intercalator - functionalized nucleotides forming the energetic hotspots , and the pseudocomplementary base pairs between 2,6-diaminopurine and 2-thiouracil need to be spatially separated in order to harness their full benefits . accordingly , a series of 13-mer dsx - modified invader probes were designed , i.e. , double - stranded probes featuring energetic hotspots comprised of conventional invader building block x , along with pseudocomplementary base pairs between regular 2,6-diaminopurine d and 2-thiothymine s monomers . the following probes were synthesized : ( i ) two different dsx - modified invader probes , in which the energetic hotspot either is next to or one nucleotide away from two pseudocomplementary base pairs ( dsx1:dsx2 and dsx3:dsx4 ) , ( ii ) an invader probe comprised of regular x monomers ( x5:x6 ) , ( iii ) an invader probe with a single pseudocomplementary energetic hotspot ( dy5:dy6 ) , and ( iv ) three pcdna probes , each containing two differentially spaced , regular pseudocomplementary base pairs ( sd1:sd2 , sd3:sd4 and sd5:sd6 ) ( table 3 ) . first , tm s for duplexes between individual probe strands and cdna ( table 3 , first two tm columns ) or crna ( table s9 ) were determined . in line with observations from the 9-mer series , x - modified ons form highly thermostable duplexes with cdna ( tm = 1112 c ) and less thermostable duplexes with crna ( tm = 3 c ) . in comparison , ons with dy motifs display slightly higher crna affinity ( tm for dy5/dy6 = 4.55.0 c , table s9 ) and slightly lower cdna affinity ( tm = 10 c , table 3 ) , again suggesting that the normally stabilizing base - pairs between 2,6-diaminoadenosine : thymine and 2-thiouracil : adenine are weakened by proximal intercalation of the pyrene moieties . encouragingly , dsx - modified ons display significantly increased cdna and crna affinity relative to x - modified ons ( tm , dna = 1415 c , table 3 ; tm , rna = 7.08.5 c , table s9 ) , suggesting that separation of the intercalators and the modified nucleobases is beneficial for binding affinity . in comparison , regular pcdna strands form far less stable duplexes with cdna ( tm , dna = 2.54.0 c , table 3 ) . benchmark invader probe x5:x6 and dy5:dy6 are both relatively thermolabile ( tm = 0.5 c and 1.5 c , respectively , table 3 ) . dsx probes are slightly less stable ( tm = 2.0 c and 3.0 c for dsx1:dsx2 and dsx3:dsx4 , respectively , table 3 ) . however , comparison with the corresponding pcdna probes suggests that the full destabilizing effect of the pseudocomplementary base pairs still is not fully realized with these probe architectures ( e.g. , compare tm of 8.0 , 0.5 , and 3.0 for sd5:sd6 , x5:x6 and dsx3:dsx4 , respectively , table 3 ) . the above tm - based conclusions are substantiated by the gibbs free energies for formation of duplexes ( table 3 ) . as a result of these stability trends , dsx probes are significantly more thermodynamically activated for dsdna - recognition than x5:x6 , dy5:dy6 , or any of the regular pcdna ( trend in grec293 values : dsx3:dsx4 < dsx1:dsx2 x5:x6 < dy5:dy6 sd5:sd6 < sd3:sd4 < sd1:sd2 , table 3 ) . tm = change in tm relative to reference duplexes dna3:dna4 ( tm 37.5 c ) , where dna3 : 5-ggta tatata ggc , dna4 : 3-ccat atatat ccg . x , y , d and s , see figure 1 . on the basis of the observed grec293 values , we decided to evaluate the dsdna - targeting efficiency of dsx1:dsx2 , dsx3:dsx4 , x5:x6 , and dy5:dy6 using a similar electrophoretic mobility shift assay as used in the 9-mer series ( figure 3a ) . thus , a dig - labeled dna hairpin ( dh8)comprised of a 13-mer double - stranded mixed - sequence stem that is linked by a t10 loop was used as a model dsdna target . incubation of dh8 with the various 13-mer invader probes results in dose - dependent formation of a slower moving band on nondenaturing page gels ( figure 3b ) . as expected from the initial 9-mer studies , the parent invader x5:x6 recognizes dh8 more effectively at low probe concentrations than dy5:dy6 , which has a pseudocomplementary energetic hotspot ( figure 3c ) . gratifyingly , invader probes with separated pseudocomplementary base pairs and energetic hotspots display improved recognition efficiency ( see curves for dsx1:dsx2 and dsx3:dsx4 , respectively , figure 3c ) , which follows the observed trend in grec293 values . it is particularly noteworthy that the use of as little as 1.0 mol equiv of dsx3:dsx4 results in 20% recognition of dh8 , especially when considering that optimal invader design normally calls for incorporation of multiple energetic hotspots . this suggests that spatial separation of pseudocomplementary base pairs and energetic hotspots is a promising principle for the design of efficient dsdna - targeting invader probes . in the present study , although there is an absence of suitable pseudocomplementary base pairs for cg - steps , we have previously shown that energetic hotspots can be comprised of intercalator - functionalized monomers that are based on any of the natural nucleobases ( although there is a preference for pyrimidine monomers ) . it is , therefore , likely possible to design dsx - like probes against dna targets with a higher gc - content than studied herein . recognition of dsdna model target dh8 using different invader probes . ( a ) illustration of recognition process ; ( b ) representative electrophoretograms for recognition of dh8 using 1- to 500-fold excess of x5:x6 , dy5:dy6 dsx1:dsx2 , or dsx3:dsx4 ; ( c ) dose response curves ( average of at least three independent experiments , error bars represent standard deviation ) . the sequence of dna hairpin dh8 is shown in figure 4 . for experimental conditions , lastly , binding specificity was studied by incubating the invader probes with dna hairpins dh9dh14 , which differ in the nucleotide sequence at one position in the stem region relative to the probes ( figure 4a ) . excellent discrimination of the nontarget dna hairpins is observed , even when using a 200-fold molar excess of x5:x6 , dy5:dy6 or dsx1:dsx2 ( figure 4b ) . similarly , high - affinity invader probe dsx3:dsx4 only results in trace recognition of dh10 and dh14 . to gain further insight into the underlying reasons for the excellent binding fidelity , we evaluated individual probe strands against singly mismatched single - stranded dna targets ( tables s10s12 ) . excellent mismatch discrimination is generally observed . in agreement with the observations from the dsdna - recognition experiments , the discrimination is least efficient when dsx3 or dsx4 are hybridized with single - stranded targets that have a sequence corresponding to the target region in dh10 and dh14 ( tm s only reduced by 57 c , relative to matched duplexes , table s12 ) . the binding of invader probes to noncomplementary dsdna targets would accordingly entail formation of two mismatched and destabilized probe - target duplexes , which is energetically prohibitive in most cases . moreover , like other structured probes , the double - stranded invader probes are also likely to exhibit improved binding specificity due to a thus , pseudocomplementary invader probes allow for strong and highly specific binding to mixed - sequence dsdna target regions at ionic conditions . ( a ) sequences and thermal denaturation temperatures of dna hairpins with isosequential ( dh8 ) or nonisosequential stems ( dh9dh14 ) ; underlined nucleotides denote sequence deviations relative to invader probes . ( b ) representative gel electrophoretograms illustrating incubation of dh8dh14 with 200-fold molar excess of x5:x6 , dy5:dy6 , dsx1:dsx2 , or dsx3:dsx4 . for experimental conditions , see figure 2 . a synthetic route to 2-n-(pyren-1-yl)methyl-2-amino-2-deoxy-2-n - methyl-2-thiouridine phosphoramidite 6 has been developed ( 10% overall yield over 13 steps from uridine ) . ons modified with the corresponding y monomer display high affinity toward cdna ( tm up to + 11.5 c ) . incorporation of 2-aminoadenosine monomer d next to the y monomer further increases cdna affinity . i.e. , 5-yd-3:3-dy-5 cassettes ) are thermolabile and activated for recognition of dsdna targets , but less so than regular invader probes with hotspots comprised of 2-n-(pyren-1-yl)methyl-2-n - methyl-2-aminouridine monomer x. in other words , close proximity of the two structural elements that normally activate pseudocomplementary dna and invader probes for dsdna - recognition i.e . , the weak base pairs between 2,6-diaminopurine and 2-thiouracil , and the destabilizing + 1 interstrand zipper arrangements of intercalating pyrene moieties does not provide a clear design benefit . in contrast , dsx probes , in which the two destabilizing structural motifs are separated , are strongly activated for recognition of mixed - sequence dsdna targets . this was confirmed in studies with model dsdna targets as efficient recognition was demonstrated to occur with excellent specificity . thus , the use of chimeric pseudocomplementary invader probes presents itself as a promising strategy for mixed - sequence recognition of dsdna for applications in molecular biology and nucleic acid diagnostics , especially since we have recently demonstrated that conventional invader probes can recognize target regions in chromosomal dna . nucleoside 1(32 ) ( 3.40 g , 4.39 mmol ) was coevaporated with anhydrous 1,2-dichloroethane ( 2 30 ml ) , redissolved in anhydrous pyridine ( 55 ml ) and cooled to 0 c . to this was added 4-dimethylaminopyridine ( dmap , 55 mg , 0.44 mmol ) and acetic anhydride ( 1.25 ml , 13.18 mmol ) . after stirring at ambient temperature for 12 h , the reaction mixture was diluted with etoac ( 150 ml ) and washed with h2o ( 80 ml ) and saturated aqueous nahco3 ( 80 ml ) . the organic layer was evaporated to near dryness and coevaporated with absolute etoh : toluene ( 2:1 v / v , 3 30 ml ) . the resulting residue ( 3.5 g ) was suspended in acoh : h2o ( 4:1 v / v , 55 ml ) and the reaction mixture was stirred overnight at room temperature . solvents were evaporated off and the resulting residue was purified by silica gel column chromatography ( 05% meoh / ch2cl2 , v / v ) to afford o5-hydroxy derivative ( 1.8 g ) as a white solid material . this material was coevaporated with anhydrous pyridine : ch2cl2 ( 1:1 v / v , 2 30 ml ) , redissolved in anhydrous pyridine : ch2cl2 ( 1:1 v / v , 44 ml ) and cooled to 20 c ( ice / salt ) . methanesulfonyl chloride ( 0.75 ml , 9.62 mmol ) was added dropwise over 30 min and the reaction mixture was stirred at 20 c for 2 h more . the reaction mixture was diluted with ch2cl2 ( 80 ml ) and washed with saturated aqueous nahco3 ( 50 ml ) . the aqueous layer was back - extracted with ch2cl2 ( 3 15 ml ) and the organic layers were combined and evaporated to dryness , followed by coevaporation with absolute etoh : toluene ( 2:1 v / v , 3 30 ml ) . the resulting residue was purified by silica gel column chromatography ( 03% meoh / ch2cl2 , v / v ) to afford nucleoside 2 ( 1.43 g , 55% ) as a white foam . rf = 0.4 ( 5% meoh in ch2cl2 , v / v ) ; maldi - hrms m / z 614.1600 ( [ m + na ] , c30h29n3o8sna , calc . 614.1568 ) ; h nmr ( 500 mhz , dmso - d6 ) 11.47 ( d , 1h , ex , j = 2.2 hz , nh ) , 8.338.36 ( d , 1h , j = 9.0 hz , py ) , 8.258.29 ( ap t , 2h , j = 7.6 hz , py ) , 8.21 ( d , 1h , j = 7.5 hz , py ) , 8.15 ( br s , 2h , py ) , 8.108.12 ( d , 1h , j = 9.0 hz , py ) , 8.048.08 ( t , 1h , j = 7.6 hz , py ) , 7.92 ( d , 1h , j = 7.5 hz , py ) , 7.73 ( d , 1h , j = 8.2 hz , h6 ) , 6.40 ( d , 1h , j = 7.4 hz , h1 ) , 5.67 ( dd , 1h , j = 8.2 hz , 2.2 hz , h5 ) , 5.39 ( dd , 1h , j = 6.6 hz , 3.6 hz , h3 ) , 4.444.48 ( m , 2h , h5 ) , 4.344.43 ( m , 3h , ch2py , h4 ) , 3.84 ( ap t , 1h , j 7.5 hz , h2 ) , 3.20 ( s , 3h , ch3so2 ) , 2.34 ( s , 3h , ch3n ) , 2.13 ( s , 3h , ch3co ) ; c nmr ( 125 mhz , dmso - d6 ) 169.7 , 162.7 , 150.5 , 140.6 ( c6 ) , 131.9 , 130.7 , 130.3 , 130.2 , 129.1 , 127.7 ( py ) , 127.3 ( py ) , 127.0 ( py ) , 126.8 ( py ) , 126.1 ( py ) , 125.1 ( py ) , 124.4 ( py ) , 124.2 , 123.8 , 123.7 ( py ) , 102.6 ( c5 ) , 83.7 ( c1 ) , 80.0 ( c4 ) , 71.7 ( c3 ) , 69.1 ( c5 ) , 64.9 ( c2 ) , 57.5 ( ch2py ) , 37.7 ( ch3n ) , 36.8 ( ch3so2 ) , 20.9 ( ch3co ) . nucleoside 2 ( 0.77 g , 1.30 mmol ) was dried through coevaporation with anhydrous 1,2-dichloroethane ( 3 6 ml ) and suspended in anhydrous etoh ( 25 ml ) . to this was added nahco3 ( 275 mg , 3.25 mmol ) and the reaction mixture was refluxed for 4 days . ch2cl2 ( 100 ml ) was added and the precipitate was filtered off and washed with ch2cl2 . the combined organic layers were evaporated to dryness and the resulting residue was purified via silica gel column chromatography ( 07% meoh in ch2cl2 , v / v ) to afford nucleoside 3 ( 0.34 g , 52% ) as a white foam . rf = 0.3 ( 10% meoh in ch2cl2 , v / v ) ; maldi - hrms m / z 522.1985 ( [ m + na ] , c29h29n3o5na , calc . 522.1999 ) ; h nmr ( 500 mhz , dmso - d6 ) 8.398.42 ( d , 1h , j = 9.3 hz , py ) , 8.248.28 ( m , 2h , py ) , 8.198.22 ( d , 1h , j = 8.0 hz , py ) , 8.14 ( br s , 2h , py ) , 8.038.10 ( d+t , 2h , py ) , 7.967.99 ( d , 1h , j = 8.0 hz , py ) , 7.93 ( d , 1h , j = 7.7 hz , h6 ) , 6.35 ( d , 1h , j = 8.5 hz , h1 ) , 5.83 ( d , 1h , j = 7.7 hz , h5 ) , 5.53 ( d , 1h , ex , j = 4.9 hz , 3oh ) , 5.12 ( t , 1h , ex , j = 5.2 hz , 5oh ) , 4.434.51 ( m , 3h , ch2py , h3 ) , 4.124.27 ( m , 2h , och2ch3 ) , 3.994.01 ( m , 1h , h4 ) , 3.583.62 ( m , 2h , h5 ) , 3.47 ( dd , 1h , j = 8.5 hz , 5.2 hz , h2 ) , 2.36 ( s , 3h , ch3n ) , 1.06 ( t , 3h , j = 7.1 hz , ch3ch2o ) ; c nmr ( 125 mhz , dmso - d6 ) 169.3 , 155.0 , 138.1 ( c6 ) , 132.7 , 130.7 , 130.3 , 130.1 , 129.0 , 127.5 ( py ) , 127.3 ( py ) , 126.9 ( py ) , 126.8 ( py ) , 126.1 ( py ) , 125.02 ( py ) , 124.99 ( py ) , 124.4 ( py ) , 124.1 , 123.8 , 123.6 ( py ) , 108.4 ( c5 ) , 87.6 ( c4 ) , 85.0 ( c1 ) , 71.2 ( c3 ) , 68.8 ( c2 ) , 64.2 ( och2ch3 ) , 61.7 ( c5 ) , 57.1 ( ch2py ) , 39.0 ( ch3n overlap with dmso - d6 signal ) , 13.6 ( ch3ch2o ) . nucleoside 3 ( 0.32 g , 0.63 mmol ) was coevaporated with anhydrous 1,2-dichloroethane ( 2 5 ml ) and redissolved in anhydrous pyridine ( 6 ml ) . to this was added dmtrcl ( 0.26 g , 0.76 mmol ) and 4-dimethylaminopyridine ( dmap , 8 mg , 0.06 mmol ) and the reaction mixture was stirred at ambient temperature for 14 h at which point it was diluted with chcl3 ( 80 ml ) and washed with saturated aqueous nahco3 ( 30 ml ) and h2o ( 30 ml ) . the aqueous layer was back - extracted with chcl3 ( 3 15 ml ) and the combined organic layers were dried ( na2so4 ) and evaporated to dryness . the resulting residue was purified by silica gel column chromatography ( 02.5% meoh in chcl3 , v / v ) to afford nucleoside 4 ( 0.45 g , 88% ) as a pale orange foam . rf = 0.7 ( 10% meoh in ch2cl2 , v / v ) ; maldi - hrms m / z 824.3319 ( [ m + na ] , c50h47n3o7na , calc . 824.3306 ) ; h nmr ( 500 mhz , dmso - d6 ) 8.388.41 ( d , 1h , j = 9.3 hz , py ) , 8.258.29 ( m , 2h , py ) , 8.178.19 ( d , 1h , j = 7.8 hz , py ) , 8.118.15 ( 2d , 2h , j = 9.1 hz , 9.1 hz , py ) , 8.048.09 ( m , 2h , py ) , 7.988.00 ( d , j = 7.8 hz , py ) , 7.69 ( d , 1h , j = 7.8 hz , h6 ) , 7.197.38 ( m , 9h , dmtr ) , 6.836.88 ( m , 4h , dmtr ) , 6.32 ( d , 1h , j = 7.9 hz , h1 ) , 5.605.64 ( m , 2d , 1 ex , j = 7.8 hz , 5.0 hz , h5 , 3oh ) , 4.484.54 ( m , 2h , ch2py ) , 4.444.48 ( m , 1h , h3 ) , 4.104.24 ( m , 3h , h4 , och2ch3 ) , 3.71 ( s , 3h , ch3o ) , 3.70 ( s , 3h , ch3o ) , 3.523.58 ( dd , 1h , j = 7.9 hz , 5.8 hz , h2 ) , 3.313.35 ( dd , 1h , j = 10.5 hz , 5.0 hz , h5 - partial overlap with h2o ) , 3.153.20 ( dd , 1h , j = 10.5 hz , 3.5 hz , h5 ) , 2.43 ( s , 3h , ch3n ) , 1.05 ( t , 3h , j = 7.0 hz , ch3ch2o ) ; c nmr ( 125 mhz , dmso - d6 ) 169.2 , 158.09 , 158.08 , 154.9 , 144.5 , 138.1 ( c6 ) , 135.3 , 135.0 , 132.6 , 130.7 , 130.2 , 130.1 , 129.7 ( dmtr ) , 129.6 ( dmtr ) , 129.0 , 127.8 ( dmtr ) , 127.6 ( dmtr ) , 127.5 ( py ) , 127.3 ( py ) , 126.91 ( py ) , 126.86 ( py ) , 126.7 ( dmtr ) , 126.1 ( py ) , 125.0 ( py ) , 124.4 ( py ) , 124.1 , 123.9 , 123.6 ( py ) , 113.20 ( dmtr ) , 113.17 ( dmtr ) , 108.1 ( c5 ) , 85.9 , 85.7 ( c4 ) , 85.3 ( c1 ) , 71.0 ( c3 ) , 68.1 ( c2 ) , 64.2 ( och2ch3 ) , 64.0 ( c5 ) , 57.1 ( ch2py ) , 55.0 ( ch3o ) , 38.8 ( ch3n ) , 13.6 ( ch3ch2o ) . a trace impurity of chcl3 was identified in the c nmr at 79.1 ppm . an ice - cold solution of anhydrous 1,1,3,3-tetramethylguanidine ( tmg , 0.68 ml , 5.42 mmol ) in anhydrous pyridine ( 10 ml ) was saturated with hydrogen sulfide gas for 1 h while maintaining the temperature at 0 c . the solution was transferred , using an argon - flushed syringe , to a precooled flask containing nucleoside 4 ( 0.44 g , 0.54 mmol ) and the reaction mixture was allowed to reach room temperature . after stirring under an argon atmosphere for 72 h , etoac ( 100 ml ) was added and the organic layer was washed with saturated aqueous nahco3 ( 50 ml ) and h2o ( 50 ml ) . the aqueous layer was back - extracted with ch2cl2 ( 3 20 ml ) and the combined organic layers were evaporated to dryness and coevaporated with absolute etoh : toluene ( 2:1 v / v , 3 15 ml ) . the resulting residue was purified by silica gel column chromatography ( 070% etoac in petroleum ether , v / v ) to afford nucleoside 5 ( 0.35 g , 82% ) as a white foam . rf = 0.8 ( 80% etoac in petroleum ether , v / v ) ; maldi - hrms m / z 812.2765 ( [ m + na ] , c48h43n3o6sna , calc . 812.2797 ) ; h nmr ( 500 mhz , dmso - d6 ) 12.77 ( br s , 1h , ex , nh ) , 8.498.52 ( d , 1h , j = 9.3 hz , py ) , 8.248.29 ( m , 2h , py ) , 8.178.20 ( d , 1h , j = 8.0 hz , py ) , 8.128.16 ( 2d , 2h , j = 9.1 hz , 9.1 hz , py ) , 8.028.11 ( m , 3h , py ) , 7.75 ( d , 1h , j = 8.2 hz , h6 ) , 7.207.39 ( m , 10h , dmtr , h1 ) , 6.846.89 ( m , 4h , dmtr ) , 5.62 ( dd , 1h , j = 8.2 hz , 1.7 hz , h5 ) , 5.55 ( d , 1h , ex , j = 4.9 hz , 3oh ) , 4.534.57 ( d , 1h , j = 13.0 hz , ch2py ) , 4.434.51 ( m , 2h , ch2py , h3 ) , 4.084.12 ( m , 1h , h4 ) , 3.71 ( s , 3h , ch3o ) , 3.70 ( s , 3h , ch3o ) , 3.483.52 ( m , 1h , h2 ) , 3.333.37 ( dd , 1h , j = 10.5 hz , 5.0 hz , h5 ) , 3.183.22 ( dd , 1h , j = 10.5 hz , 3.0 hz , h5 ) , 2.44 ( s , 3h , ch3n ) ; c nmr ( 125 mhz , dmso - d6 ) 176.6 , 159.0 , 158.11 , 158.10 , 144.5 , 140.8 ( c6 ) , 135.3 , 135.0 , 132.6 , 130.7 , 130.3 , 130.2 , 129.73 ( dmtr ) , 129.68 ( dmtr ) , 129.2 , 128.0 ( py ) , 127.9 ( dmtr ) , 127.6 ( dmtr ) , 127.3 ( py ) , 127.0 ( py ) , 126.8 ( py ) , 126.7 ( dmtr ) , 126.1 ( py ) , 125.02 ( py ) , 125.00 ( py ) , 124.4 ( py ) , 124.1 , 124.0 ( py ) , 123.9 , 113.3 ( dmtr ) , 113.2 ( dmtr ) , 106.9 ( c5 ) , 88.0 ( c1 ) , 86.0 , 85.4 ( c4 ) , 71.0 ( c3 ) , 68.6 ( c2 ) , 63.9 ( c5 ) , 57.6 ( ch2py ) , 55.0 ( ch3o ) , 39.2 ( ch3n ; overlap with dmso - d6 ) . to a flame - dried round - bottomed flask containing nucleoside 5 ( 150 mg , 0.19 mmol ) was added anhydrous ch2cl2 ( 2 ml ) , anhydrous n , n - diisopropylethylamine ( dipea , 165 l , 0.95 mmol ) and 2-cyanoethyl - n , n - diisopropylchlorophosphoramidite ( pcl reagent , 85 l , 0.38 mmol ) . the reaction mixture was stirred at room temperature for 3.5 h at which point ice - cold etoh ( 1.0 ml ) was added . the reaction mixture was evaporated to dryness and the resulting residue was purified by silica gel column chromatography ( 055% etoac in petroleum ether , v / v ) followed by precipitation from cold petroleum ether to afford nucleoside 6 ( 153 mg , 81% ) as a white foam . rf = 0.6 ( 50% etoac in petroleum ether , v / v ) ; maldi - hrms m / z 1012.3843 ( [ m + na ] , c57h60n5o7psna , calc . 1012.3887 ) ; p nmr ( 121 mhz , cdcl3 ) 150.9 , 149.6 . modified ons were synthesized on a 0.2 mol scale using a dna synthesizer , succinyl - linked lcaa - cpg ( long chain alkyl amine controlled pore glass ) columns with a pore size of 500 , and standard protocols for incorporation of a , c , g and t dna phosphoramidites . the following hand - coupling conditions were used for incorporation of the corresponding phosphoramidites of monomers x , y , s ( n3/o4-toluoyl protected ) and d ( bis(diisobutylaminomethylidene)-protected ) ( coupling time ; activator ; coupling yield ) : x ( 15 min ; 5-[3,5-bis(trifluoromethyl)phenyl]-1h - tetrazole ; 99% ) , y ( 15 min ; 4,5-dicyanoimidazole ; 95% ) and s / d ( 15 min ; 4,5-dicyanoimidazole ; 99% ) . modified phosphoramidites were used at 50-fold molar excess and 0.05 m concentration in ch3cn . extended oxidation ( 45 s ) with standard 0.05 m aqueous iodine was used for d1d4 and x1x6 . extended oxidation ( 2 5 min oxidation with an acetonitrile wash between oxidations ) using a tert - butylhydroperoxide / ch3cn / h2o solution ( 10/87/3 , v / v / v ) was used for all ons containing s and y modifications to prevent desulfurization . cleavage from solid support and removal of protecting groups was accomplished by treatment with 32% aq . ons were purified in the dmt - on mode via ion - pair reverse phase hplc ( c18 column ) using a 0.05 m triethylammonium acetate . acoh ) and precipitation ( naoac / naclo4/acetone , 18 c for 1216 h ) . the identity of synthesized ons was established through maldi - ms analysis ( table s1 ) recorded in positive ions mode on a quadrupole time - of - flight tandem mass spectrometer equipped with a maldi source using anthranilic acid , 3-hydroxypicolinic acid ( 3-hpa ) or 2,4,6-trihydroxyacetophenone ( thap ) as matrices . purity was verified by ion - pair reverse phase hplc running in analytical mode ( > 90% unless otherwise mentioned ) . on concentrations were estimated using the following extinction coefficients for dna ( od/mol ) : g ( 12.01 ) , a ( 15.20 ) , t ( 8.40 ) , c ( 7.05 ) ; rna ( od/mol ) : g ( 13.70 ) , a ( 15.40 ) , u ( 10.00 ) , c ( 9.00 ) ; pyrene ( 22.4),d ( 8.5 ) , s ( 10.0 ) and y ( 32.4 ) . strands were thoroughly mixed and denatured by heating to 7085 c , followed by cooling to the starting temperature of the experiment . thermal denaturation temperatures ( tm s ) of duplexes ( 1.0 m final concentration of each strand ) were measured using a uv / vis spectrophotometer equipped with a 12-cell peltier temperature controller and determined as the maximum of the first derivative of thermal denaturation curves ( a260 vs t ) recorded in medium salt phosphate buffer ( tm buffer : 100 mm nacl , 0.1 mm edta and ph 7.0 adjusted with 10 mm na2hpo4 and 5 mm na2hpo4 ) . the temperature of the denaturation experiments ranged from at least 15 c below tm to 20 c above tm ( although not below 3 c ) . thermodynamic parameters for duplex formation were determined through fitting of baselines of denaturation curves ( vant hoff analysis ) using software provided with the uv / vis spectrometer . bimolecular reactions , two - state melting behavior , and a heat capacity change of cp = 0 upon hybridization were assumed . a minimum of two experimental denaturation curves were each analyzed at least three times to minimize errors arising from baseline choice . uv vis absorption spectra ( range 200600 nm ) were recorded at 10 c using the same samples and instrumentation as in the thermal denaturation experiments . steady - state fluorescence emission spectra of y- or dy - modified ons and the corresponding duplexes with complementary dna / rna targets , were recorded in nondeoxygenated thermal denaturation buffer ( each strand at 1.0 m concentration ) and obtained as an average of five scans using an excitation wavelength of ex = 350 nm . excitation and emission slits of 5.0 and 2.5 nm , respectively , were used along with a scan speed of 600 nm / min . experiments were determined at 5 c ( to ascertain maximal hybridization of probes to dna / rna targets ) under n2 flow ( to prevent condensation ) . unmodified dna hairpins dh1dh14 were obtained from commercial sources and used without further purification . the dna hairpins were 3-dig - labeled using the second generation dig gel shift kit ( roche applied bioscience ) following the manufacturer s recommendation . dig - labeled ons obtained in this manner were diluted and used without further purification in the recognition experiments . preannealed probes ( 85 c for 10 min , cooled to room temperature over 15 min ) and dig - labeled dna hairpins ( 34.4 nm ) were mixed and incubated in hepes buffer ( 50 mm hepes , 100 mm nacl , 5 mm mgcl2 , 10% sucrose , 1.44 mm spermine tetrahydrochloride , ph 7.2 ) for the specified time at ambient temperature ( 21 3 c ) . the reaction mixtures were then diluted with 6x dna loading dye ( fermentas ) and loaded onto a 16% nondenaturing polyacrylamide gel . electrophoresis was performed using a constant voltage of 70 v for 2.5 h at 4 c using 0.5 tbe as a running buffer ( 45 mm tris , 45 mm boric acid , 1 mm edta ) . gels were blotted onto positively charged nylon membranes ( roche applied bioscience ) using constant voltage with external cooling ( 100 v , 4 c ) . the membranes were exposed to antidigoxigenin - ap fab fragments as recommended by the manufacturer of the dig gel shift kit , transferred to a hybridization jacket , and incubated with the substrate ( cspd ) in detection buffer for 10 min at 37 c . the chemiluminescence of the formed product was captured on x - ray film , which was developed using an x - omatic 1000a x - ray film developer ( kodak ) . the efficiency of dna recognition was determined as the intensity ratio between the recognition complex band and the total lane . nonlinear regression was used to fit data points from dose response experiments , using a script written for the solver module in microsoft office excel . the following nomenclature describes the relative arrangement between two pyrene - functionalized monomers positioned on opposing strands in a duplex : the number n describes the distance measured in number of base pairs and has a positive value if a monomer is shifted toward the 5-side of its own strand relative to a second reference monomer on the other strand . conversely , n has a negative value if a monomer is shifted toward the 3-side of its own strand relative to a second reference monomer on the other strand .

the development of molecular strategies that enable recognition of specific double - stranded dna ( dsdna ) regions has been a longstanding goal as evidenced by the emergence of triplex - forming oligonucleotides , peptide nucleic acids ( pnas ) , minor groove binding polyamides , and more recently engineered proteins such as crispr / cas9 . despite this progress , an unmet need remains for simple hybridization - based probes that recognize specific mixed - sequence dsdna regions under physiological conditions . herein , we introduce pseudocomplementary invader probes as a step in this direction . these double - stranded probes are chimeras between pseudocomplementary dna ( pcdna ) and invader probes , which are activated for mixed - sequence dsdna - recognition through the introduction of pseudocomplementary base pairs comprised of 2-thiothymine and 2,6-diaminopurine , and + 1 interstrand zipper arrangements of intercalator - functionalized nucleotides , respectively . we demonstrate that certain pseudocomplementary invader probe designs result in very efficient and specific recognition of model dsdna targets in buffers of high ionic strength . these chimeric probes , therefore , present themselves as a promising strategy for mixed - sequence recognition of dsdna targets for applications in molecular biology and nucleic acid diagnostics .

Introduction Results and Discussion Conclusion Experimental Section

PMC4499986

the human foot is composed of three arch structures , namely the medial longitudinal arch ( mla ) , lateral longitudinal arch , and transverse arch . these arch structures reduce pressure on the soft tissue by dispersing the loading stress and increasing the efficiency of force transmission by functioning as a spring during walking1 . the mla has been measured to evaluate the role of arch structure in a physical therapy setting , with the navicular bone being regarded as a landmark of the arch apex , as it is palpable and deviates minimally . some methods of mla measurement have been previously described , including navicular drop ( nd ) , in which the difference in navicular height between the standing and sitting positions is measured2 . the height and general shape of the mla are maintained by the plantar fascia , ligaments , and intrinsic and extrinsic muscles of the foot3 . previous studies have reported that nd is related to lower limb disability4 , 5 and that it can be decreased after toe exercise6 ; taken together , these findings suggest that toe muscle strength may relate to arch structure and lower limb disability . the extrinsic muscles running across the ankle joint include the flexor hallucis longus ( fhl ) and flexor digitorum longus . on the other hand , the intrinsic muscles do not run across the ankle joint and do not contribute to ankle movement . both the intrinsic and extrinsic muscles are stretched by dorsiflexion of the metatarsophalangeal ( mtp ) joints , whereas only the extrinsic muscles are relaxed by plantar flexion of the ankle joint7 . the exhibited maximal torque is affected by the joint angle8 , and a relationship between joint angle and plantar flexion torque of the mtp joints has been reported previously9 . thus , these previous studies suggest that excessive nd can cause lower limb disability and that this can be controlled by toe exercise . however , the dynamometer used in the previous studies measured only toe grip strength , i.e. , the plantar flexion force of all toes at the plantar flexion position of the mtp joint10 . moreover , the relationship between nd and the measurement position of plantar flexion torque of the mtp joints has not been studied . therefore , the aims of this study were to clarify the relationship between nd and plantar flexion torque of the first and second - fifth mtp joints and to determine the differences between neutral positions of the mtp and ankle joints and stretching position of the intrinsic muscles . in a previous study , the mla was found to be raised by contraction of the intrinsic foot muscles11 . accordingly , we hypothesized that the relationship between nd and plantar flexion torque of the mtp joints would change with different measuring positions , and we believe that the results of this study will provide valuable information for exercise and risk management to prevent lower limb disabilities . the mean values standard deviations ( sds ) for their age , height , and body weight were 23.8 1.5 years , 1.69 0.04 m , and 58.4 8.1 kg , respectively . this study was approved by the ethics committee on human research of waseda university ( approval number : 2013 - 195 ) . the authors provided all subjects with an explanation of the purpose and methods of this study and details of the study protocol , and written informed consent was obtained from all subjects . navicular height was defined as the distance from the floor to the tubercle of the navicular bone , as measured with a caliper . palpation of the tubercle of the navicular bone was performed by a physical therapist who had more than 3 years of experience in orthopedic physical therapy . nd was calculated by subtracting the navicular height in the standing position from that in the sitting position . a custom - made mtp joint plantar flexion torque meter device was used to measure mtp joint plantar flexion torque . the subjects were seated on a chair , with their trunk , thighs , lower thighs , and dominant foot fastened to the chair and the torque meter device . after warming up , each subject performed maximal voluntary isometric contraction of plantar exion of the first mtp joint , followed by that of the second - fifth mtp joint for 3 s to determine the maximal voluntary contraction ( mvc ) torque . the plantar flexion torque was calculated as the tensile force of a strain gauge ( tu - br 500n , teac , japan ) multiplied by the 0.10-m lever arm of the force plate ( fig . 1.structure of the torque meter used to measure isometric plantar flexion torque at the metatarsophalangeal ( mtp ) joints . mtp joint plantar flexion torque was calculated using the following formula : plantar flexion torque ( nm ) = strain ( n ) moment arm 0.10 ( m ) . ) . we converted the tensile force data from analog to digital ( power lab , ad instruments , australia ) via an amplifier ( dpm-711b , kyowa electronics , japan ) using a personal computer . mvc torque was measured at two positions : at a neutral position of the ankle and mtp joints and at 45 dorsiflexion of the mtp joints with 20 plantar flexion of the ankle joint . structure of the torque meter used to measure isometric plantar flexion torque at the metatarsophalangeal ( mtp ) joints . mtp joint plantar flexion torque was calculated using the following formula : plantar flexion torque ( nm ) = strain ( n ) moment arm 0.10 ( m ) . further , we measured mvc torque again on a different day to determine the test - retest repeatability of these measurements , which was accomplished by calculating the mean coefficient of variance ( mean / sd ) and intraclass correlation coefficient ( icc ) . to assess the iccs , we used the criteria advocated by fleiss12 , in which an icc > 0.75 is defined as excellent reliability . a pearson correlation coefficient was used to test the correlation between the nd and mvc torques . for all tests , the mean coefficients of variance of the mvc torques of the first mtp and second - fifth mtp joints measures were 7.6% and 7.4% , respectively , and the corresponding iccs ( 1.1 ) were 0.81 and 0.87 , respectively . significant negative correlations were seen between the nd and the mvc torques at 20 plantar flexion of the ankle joint with 45 dorsiflexion of the mtp joints of the first mtp joint ( r = 0.78 , p < 0.01 ) and second - fifth mtp joints ( r = 0.82 , p < 0.01 ) ( table 1table 1.relationship between navicular drop ratio and maximal voluntary contraction torque ratio of the plantar flexion of the metatarsophalangeal jointsposition of the mtp and ankle jointsmeasured jointsmvc torque(n / kg)nds(mm)correlationcoefficientsneutralfirst mtp0.13 0.037.4 2.50.52second - fifth mtp0.09 0.030.3845 dorsiflexion of the mtp joints , 20 plantar flexion of the ankle jointfirst mtp0.17 0.030.78*second - fifth mtp0.12 0.020.82*mean sds ; mtp joint : metatarsophalangeal joint ; mvc torque : maximal voluntary contraction torque ; nd : navicular drop ; * p < 0.01 ) . however , no significant correlations were found between the nd and mvc torques at the neutral position of the ankle and first mtp joint ( r = 0.52 , p > 0.05 ) and second - fifth mtp joints ( r = 0.38 , p > 0.05 ) ( table 1 ) . mean sds ; mtp joint : metatarsophalangeal joint ; mvc torque : maximal voluntary contraction torque ; nd : navicular drop ; * p < 0.01 the iccs ( 1.1 ) for both the first and second - fifth mtp joints were more than 0.75 in this study and showed excellent reliability12 , and the test - retest repeatability of the measurements of mvc torque were confirmed . significant negative correlations were observed between nd and mvc torque at 45 dorsiflexion of the mtp joints with 20 plantar flexion of the ankle joint . on the other hand , there was no significant correlation between nd and mvc torque at the neutral position , indicating that nd relates to intrinsic muscle strength . mvc torque is affected by muscle length , and muscle length in turn is known to be affected by the joint angle8,13 . in a previous study , the muscle length of the fhl was found to vary an average of 0.22 mm by 1 plantar flexion or dorsiflexion of the first mtp joint and 0.50 mm by 1 plantar flexion or dorsiflexion of the ankle joint7 . furthermore , according to the same study , at 45 dorsiflexion of the first mtp joint , the muscle length of the fhl increased approximately 10 mm from that in neutral position , whereas at 20 plantar flexion of the ankle joint , the length of the fhl decreased approximately 10 mm from that in neutral position . in the present study , the length variation of the extrinsic muscle was countered and the intrinsic muscles were lengthened by 20 plantar flexion of the ankle joint and 45 dorsiflexion of the first mtp joint . at this position , a higher torque can be exerted even though the length of the extrinsic muscle remains constant8 . in addition , it has been reported that toe flexion exercises performed during plantar flexion of the ankle joint are an effective method of intrinsic foot flexor strength training14 . according to these findings , it appears as if the intrinsic muscles are capable of exerting force in this position . furthermore , it has been reported that the mla was raised upon inducing contraction of the intrinsic muscles via electrical stimulation11 . at the neutral position of the ankle and mtp joints , both the intrinsic and extrinsic muscles can produce similar force . however , the produced force ratio of the extrinsic muscle to the intrinsic muscles is relatively large at the neutral position of the ankle and mtp joints as compared to at 20 plantar flexion of the ankle joint with 45 dorsiflexion of the mtp joints , and this is likely the reason for the lack of a significant correlation between mvc torque and nd in this condition . taken together , these results suggest that the intrinsic foot muscles contribute greatly to the construction of the mla and that nd relates to maximal plantar flexion torque of mtp joints at 20 plantar flexion of the ankle joint with 45 dorsiflexion of the first mtp joint . these results support a previous finding that nd is correlated with electromyographic activity of the abductor hallucis muscle15 . moreover , these results indicate that the strength of the intrinsic toe plantar flexion muscles is related to nd , suggesting that exercises targeting the intrinsic muscles may not only help improve excessive nd but also prevent sport - related disorders caused by excessive nd . the main limitation of this study was that the mla was affected not only by the toe flexor muscle but also by the muscle that did not contribute to mtp joint plantar flexion ( e.g. , the tibialis posterior and peroneus longus muscles ) . furthermore , we did not investigate extrinsic muscle length according to variations of the mtp joint angle . therefore , we instead discussed the results based on the length variation of the fhl . future studies aimed at assessing the flexor digitorum longus length according to variations of the second to fifth mtp joints are needed . in conclusion , we found that nd relates to mtp joint muscle strength in plantar flexion where the intrinsic toe plantar flexion muscles are capable of exerting force . this result suggests that exercises targeting the intrinsic muscles may help improve excessive nd .

[ purpose ] the purpose of this study was to determine the relationship between navicular drop and plantar flexion torque of the first and second - fifth metatarsophalangeal joints . [ subjects ] ten healthy young men participated in this study . [ methods ] the pearson product - moment correlation coefficient was calculated to determine the relationship between navicular drop and plantar flexion torque of the first and second - fifth metatarsophalangeal joints . [ results ] significant negative correlations were observed between navicular drop and plantar flexion torques in the lengthened position of the intrinsic toe plantar flexion muscles , but no correlations were found between navicular drop and plantar flexion torques in the neutral position of the ankle and metatarsophalangeal joints . moreover , the intrinsic toe plantar flexion muscles were found to contribute to the formation of the medial longitudinal arch . [ conclusion ] navicular drop correlates with metatarsophalangeal joint muscle strength in plantar flexion where the intrinsic toe muscles are capable of exerting force .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC5430289

stress fractures occur due to repetetive loading of the bones with submaximal stress rather than a single traumatic event . they can occur in all bones of the lower extremity , particularly in people predisposed to repetetive strain , such as athletes . any sports involving repetitive jumping , cutting , landing , long distance running , tennis , basketball may predispose the athlete to stress fractures . the incidence increases with a sudden increase in intensity or duration of training , poor physical conditioning , or incorrect footwear . excessive forefoot or ankle varus , leg length discrepancy , a high or low longitudinal arch , muscle imbalance and tightness are other risk factors . in women , repeated stress , low bone mineral density and dietary restraint have been reported as a specific risk triad1 . vitamin k promotes clotting and is involved in the activation of important proteins in blood coagulation , prothrombin ( ii ) , factor vii , factor ix , and factor x , as well as protein c and protein s. deficiency of these factors can result in defective clotting and a bleeding disorder . when vitamin k was discovered , it was believed to have only the role of supporting coagulation , and it was given the name because of low plasma concentration , vitamin k has been assessed by functional methods , using prothrombin time to determine its effect on clotting time . further research has shown that it also functions as a coenzyme that enhances calcium incorporation into bone . the currently known forms of vitamin k can be grouped into the following three categories : vitamin k1 ( phytomenadione or phytomenadione ) , vitamin k2 ( menaoquinones ) , and vitamin k3 ( menadione ) . vitamins k1 and k3 are fat - soluble , whereas vitamin k2 is water - soluble . technically , only vitamin k1 is recognized as a true vitamin , which is essential for human and animal well - being and present in fresh green vegetables such as broccoli , cabbage , lettuce , and spinach . vitamins k2 and k3 can be derived as metabolites of vitamin k1 in various tissues or the gastrointestinal tract in animals or humans . it has been shown that vitamin k2 modulates the genetic expression of collagen type 1 and osteocalcin which leads to increased produciton of these proteins by osteoblasts3 . its reducing effect on osteoblasts apoptosis4 and stimulating effect on other bone blastic proteins , such as osteoprotegerin and osteopontin5 , have been reported . regarding bone remodelling resorption , the reduction of osteoclast production from bone marrow cells6 and induction of osteoclasts apoptosis these metabolic effects are achieved through vitamin k2 s ability to downregulate inflammation ( il-6 , pge2 ) and cell proliferation while increasing apoptosis7 . it should be emphasized that this would be effective only if osteoclastic activity is excessively high . vitamin k2 may reduce the accelerated bone resorption induced by menopausal hormonal loss8 or corticosteroid treatment9 , 10 . vitamin k deficiency as a possible contributing factor for stress fractures has not been described before , although there are reports documenting its effect on osteoporotic femoral neck fractures . a 13-year - old basketball player was admitted complaining of right foot pain for the previous 5 days . he had no remarkable medical history , except for a deficiency of vitamin k when he was 3 or 4 years old . he never had any bleeding disorder and the parents did not remember why vitamin k level was checked . he had been playing basketball since he was 10 years old . his practices were 1.5 to 2 hours / day , 5 days a week . he reported no recent increase in the intensity or duration of the training sessions . he did not have pain during normal activities of daily living , but he was unable to practice because of pain . physical examination findings were tenderness of the plantar fascia and lateral side of the ankle with palpation , low medial longitudinal arches , and tight hamstrings and achilles tendons . magnetic resonance imaging showed edema of the cuboid and third cuneiform bones , together with mild effusion of the surrounding soft tissues . tendonitis of the flexor hallucis and digitorum longi were also present ( fig . 1.magnetic resonance imaging ( mri ) findings of the patient on admission showing edema of the cuboid and third cuneiform bones , mild effusion of the surrounding soft tissues , tendonitis of the flexor hallucis and digitorum longi ) . the foot was immobilized for 4 weeks and he received physiotherapy consisting of electrical stimulation ( both tens for pain relief and muscle stimulation for strengthening ) and hydrotherapy during this time . his normal activities of daily living were allowed with the immobilized foot . between the 4th and 6th weeks , he reported about 80% improvement of symptoms and partial weight bearing was allowed . he had only mild tenderness of the plantar fascia on palpation . at the end of the 6th week , he reported diffuse and more severe pain . diffuse tenderness on the dorsal and plantar surfaces of the foot was evident and ankle movements were painful and limited . updated imaging was ordered and showed increased areas of edema of both the cuboid and the third cuneiform bones and surrounding soft tissues when compared with the first imaging . there was also edema of the talar bone on the navicular side and the proximal first metatarsal bone which had not been present prior . 2.magnetic resonance imaging ( mri ) findings of the patient at 6th week showing increased areas of edema of both the cuboid and the third cuneiform bones and surrounding soft tissues , edema of the talar bone on the navicular side and the proximal first metatarsal bone , tenosynovitis of the flexor hallucis and digitorum longi ) . the test results were as follows ( nr , normal range):hb : 11.5 g / dl ( nr : 1116 ) , erythrocyte sedimentation rate ( esr ) : 33 mm / h ( nr : < 15 ) , c - reactive protein ( crp ) : 1.75 mg / dl ( nr : < 0.22 ) , rheumatoid factor ( rf ) : 8 iu/ ml ( nr : < 14 ) , antinuclear antibody ( ana ) : negative , fasting blood sugar : 104 mg / dl ( nr : 70100 ) , calcium : 10 mg / dl ( nr : 9.210.7 ) , phosphorus : 4.6 mg / dl ( nr : 3.15.3 ) , alkaline phosphatase ( alp ) : 263 u / l ( nr : 169618 ) , parathormone ( pth ) : 38 pg / ml ( nr : 1472 ) , vitamin d , 25 hydroxy : 28.2 ng / ml ( nr : 2580 ) , vitamin b12 : 592 pg / ml ( nr : 200835 ) , blood urea nitrogen ( bun ) : 9 mg / dl ( nr : 4.6722.43 ) , creatinine : 0.79 mg / dl ( nr : 0.51.1 ) , alt : 22 u / l ( nr : 1659 ) , ast : 18 u / l ( nr : 1036 ) . magnetic resonance imaging ( mri ) findings of the patient on admission showing edema of the cuboid and third cuneiform bones , mild effusion of the surrounding soft tissues , tendonitis of the flexor hallucis and digitorum longi magnetic resonance imaging ( mri ) findings of the patient at 6th week showing increased areas of edema of both the cuboid and the third cuneiform bones and surrounding soft tissues , edema of the talar bone on the navicular side and the proximal first metatarsal bone , tenosynovitis of the flexor hallucis and digitorum longi although not routinely ordered in practice , as the parents reported a nonspecific history , and the other findings did not explain the clinical situation , serum vitamin k level was ordered , with the result < 0.1 ng / ml ( nr : 0.122 ) . at the time of blood sampling for vitamin k testing , fasting blood sugar test was repeated because of the above - normal range during the first test ; the result was 88 mg / dl . dietary habits were also questioned ; an exact food diary could not be obtained , but his parents stated that he had fresh green vegetables like broccoli and lettuce on an average of 3 to 4 times per week . overall , his diet was quite balanced , with consumption of lean meat or fish , dairy products , citrus fruits on a daily basis . his foot was immobilized for an additional 2 weeks , and partial weight bearing was then allowed . during the total and partial non - weight bearing periods , he performed isometric exercise and hamstring and achilles stretches . he was encouraged to consume more fresh green vegetables for at least one meal per day . following 5 months from the beginning of the symptoms , he started practicing with longitudinal arch supports ; he started participating in games at the 6th month . stress fractures of the lower extremities are common in athletes and they are being more commonly diagnosed both by increased awareness of the physicians and by imaging techniques . gait , limb length discrepancy , and tightness of the proximal muscles and tendons should be noted in the physical examination as well as the general appearance of the foot regarding shape , deformity , swelling , pain and tenderness , and range of motion . the present case exhibited low medial longitudinal arches and tight hamstrings as well as short achilles tendons ; these were all predisposing factors for stress fractures . these factors were appropriately corrected during the treatment process and after the patient returned to sport . in general , the severity of the fracture and pain can dictate how soon an athlete can re initiate regular training . in this case , the initial expectation was to restart training with a gradual increase in intensity after 6 weeks however , this was no achieved in our case . a second magnetic resonance imaging was ordered as the patient s symptoms and physical examination findings progressed despite treatment . although this case was not bilateral , as the condition progressed , these issues were considered . esr and crp were elevated ; this was not unexpected as there was an inflammatory reaction in the body . it was rechecked in the same sample extracted to test vitamin k level , and shown to be within the normal range . serum vitamin k level is not routinely checked as it is found in very low concentrations in the serum ; instead , prothrombin time is used to determine its effect on clotting time . the patient had no history of any bleeding disorder , including no excessive bleeding after circumcision . the parents did not recall the patient s previous history of serum vitamin k level . although the previous test results were unavailable , it was reported to be low . they had not been advised to take any action on the situation . after finding the level to be lower than normal limits , we investigated his dietary habits , as vitamin k is found in green leafy vegetables such as spinach , broccoli , lettuce , and others . our patient had some of these vegetables as part of his diet , but like many of teenagers of his age , he was not motivated to consume them . poor vitamin k staus is reported to be associated with low bone mass , osteoporosis , and fracture risk in adults12,13,14 , but its relationship with stress fractures has not been reported . considering the effects of vitamin k on osteocalcin , it was concluded that the vitamin k requirement of children is much larger than that of adults because osteocalcin synthesis is at least 10-fold higher during growth than after peak bone mass has been reached15 . in an epidemiological study investigating the association of hip fracture incidence and intake of calcium , magnesium , vitamin d , and vitamin k in japan , a striking pattern of high intake of vitamin k and low incidence of hip fractures in eastern areas of japan was apparent , with the opposite pattern a low intake of vegetables rich in vitamin k and a high incidence of hip fracture in western areas16 . the observers found significant inverse associations between hip fracture incidence and each nutrient , but after adjusting for covariates , the strongest correlation was seen with vitamin k. the effects of calcium and vitamin d on bone metabolism are very well known with no doubt that individuals should have adequate intake through diet and/or supplements . the need is increased in certain conditions like adolescence , pregnancy , and post - menapausal states . calcium and vitamin d deficiency may lead to both osteoporotic fractures and stress fractures . the deficiency of vitamin k , apart from its effects on bleeding , has been linked with osteoporotic fractures ; however , it has not currently been related to the poor healing of stress fractures . in the present case , there were clear risk factors such as repetitive loading with poor mechanics resulting from low medial longitudinal arches , tight hamstrings , and achilles tendons . however , although measures to treat the condition were taken , the progress was not as expected , and progressively worsened . assessing serum vitamin k level was not initially planned until the parents reported that a previous history was present . the patient s eating habits were like most of the adolescents of his age , but he was not very strict on eating no vegetables . he cooperated when he was encouraged to increase the amount of green vegetables in his diet . the present case can not be directly related to vitamin k deficiency alone , and , as mentioned above , an unhealing stress fracture due to vitamin k deficiency has not been reported previously . the patient returned to full sport activities at the 6th month , which is the approximate time period to be expected with many types of stress fracures . thus , a claim that increasing the amount of green vegetables in his diet necessarily improved the situation is unfounded . additionally , a follow - up vitamin k level assessment was not performed and it is not known whether the level reached normal limits after the dietary changes . in conclusion , along with assessing calcium and vitamin d status , which has a very well - documented effect on bone metabolism , we advise questioning on consumption of green vegetables to gain a full picture on the potential vitamin k level in patients with stress fractures , especially if healing does not occur as expected . if a poor status is confirmed , encouraging the patient to consume green vegetables in at least one meal a day might assist in the healing process . it is not recommended to examine serum vitamin k levels in patients with stress fractures on a routine basis .

[ purpose ] to report an adolescent male basketball player with nonhealing stress fractures of the foot and discuss the probable factors . [ subject and methods ] a 13-year - old basketball player presented with right foot pain . he had been playing basketball for three years and practicing 5 days / week . he denied any increase in daily training intensity . magnetic resonance imaging confirmed stress fractures of the cuboid and cuneiform , with mild edema of the soft tissues between the tarsal bones and tenosynovitis of the flexor hallucis and flexor digitorum longi . the foot was immobilized for 4 weeks , with progressive weight bearing introduced at the fifth week . at the 6th week , while still restricted to partial weight bearing , he reported diffuse severe pain . the entire foot was painful with palpation , and new imaging showed stress fractures of the talus , cuboid , cuneiform , and proximal first metatarsal bones , and tenosynovitis of the flexor hallucis longus and flexor digitorum longus tendons with progression of the soft tissue edema around the tarsal bones . acute phase reactants were elevated ; vitamin k level was low . [ results ] he started participating in games again at the 6th month post - injury . [ conclusion ] management of patients with stress fractures includes immobilization , physical therapy , and biomechanical arrangements . if the expected healing does not occur , a deficiency of vitamin k might be considered as a factor . questioning on dietary habits of the patient and encouraging adequate intake of the deficient nutrient might assist in the healing process .

INTRODUCTION SUBJECT AND METHODS RESULTS DISCUSSION

PMC3686150

osteoporosis and the fractures that result from osteoporosis are a major public health problem worldwide . for example , it has been estimated that osteoporosis causes more than 8.9 million fractures annually , resulting in an osteoporotic fracture every 3 seconds . in the united states ( usa ) , the prevalence of osteoporosis and low bone mass includes almost 44 million women and men 50 years of age and older . , it is estimated that more than 61 million women and men in the usa will have osteoporosis or low bone mass . exercise is a nonpharmacologic intervention that has been recommended for increasing and/or maintaining bone mineral density ( bmd ) in adults [ 3 , 4 ] . however , the investigative team is not aware of any previous meta - analytic research that has focused on dropouts and compliance with respect to participants enrolled in nonbehaviorally focused randomized controlled exercise intervention programs targeting bmd in adults . this has important implications from both a research and practice perspective . from a research perspective , knowledge of these potential factors can assist trialists in developing experimental designs that minimize dropout and maximize compliance , thereby allowing one to better identify the true effects of exercise on bmd and other outcomes of interest . from a practice - based standpoint , such knowledge can aid one in determining the feasibility of exercise as a nonpharmacologic intervention for improving bmd in adults . in addition , a recent systematic review that attempted to address the determinants of exercise and physical activity participation in older adults recommended that additional research on this topic was needed . given the importance of this issue , the purpose of the current study was to use the aggregate data meta - analytic approach to examine dropouts and compliance in exercise interventions targeting bmd in adults , including selected factors associated with such . studies were included if they met the following criteria : ( 1 ) nonbehaviorally focused randomized trials with a comparative control group ( e.g. , nonintervention ) , ( 2 ) adults 18 years of age , ( 3 ) participants not engaged in a regular exercise program prior to study enrollment , ( 4 ) ground and/or joint reaction force exercise 24 weeks in which fn and/or ls bmd was assessed , ( 5 ) published and unpublished ( master 's theses and dissertations ) studies in any language since january 1989 , and ( 6 ) data available for dropouts and/or compliance to the exercise intervention . any studies not meeting all six criteria were excluded . studies were limited to randomized controlled trials in which the exercise intervention lasted at least 24 weeks because most bmd intervention studies last at least this long due to the fact that the bone remodeling process typically takes approximately 24 weeks to complete [ 6 , 7 ] . studies were limited to those that assessed fn and/or ls bmd because these are the sites most often studied given that they are the most common sites for fracture . we limited studies to those that assessed bmd because it is considered to be the best predictor for osteoporosis . the year 1989 was chosen as the start date for inclusion since it appeared to be the first time that a randomized controlled trial on exercise and bmd in adult humans was conducted . potentially eligible studies were derived from a large database that was the result of searching six electronic reference databases ( pubmed , embase , sportdiscus , cochrane central register of controlled clinical trials , cinahl , and dissertation abstracts international ) , cross - referencing , hand searching , and expert review ( dr . wendy kohrt , personal communication ) . all searches were conducted by the second author with assistance from a health sciences librarian . detailed search strategies for all databases searched are available upon request from the corresponding author . for the current meta - analysis , if consensus could not be reached , the consultant for this project provided a recommendation ( dr . wendy kohrt , personal communication ) . data from multiple reports on the same participants from the same study was avoided by only including data from the same participants once . risk of bias was assessed using the cochrane risk of bias assessment instrument . given the nature of study outcomes , assessment for risk of bias was limited to sequence generation , allocation concealment , and blinding ( participants , personnel , and outcome assessors ) . results were categorized as being at a low , high , or unclear risk for bias . the a priori primary outcomes for this study were dropouts in the exercise and control groups as well as compliance to the exercise intervention . for all three outcomes , the effect size ( es ) of choice was the proportion . dropouts were defined as the proportion of participants who dropped out of the study after being randomized to the intervention or comparative control group . compliance was defined as the proportion of required exercise sessions that the exercise participants completed . each proportion was converted to the logit event rate and its variance prior to pooling . in addition , 95% confidence intervals were generated for each mean event rate for each group from each study . . this approach weights studies by the inverse of the variance and incorporates between - study heterogeneity into the model . all analyses were conducted using the logit event rate and then back transformed to proportions for the purpose of enhancing interpretation and application . an alpha value 0.10 was considered as statistically significant heterogeneity for q while an i statistic greater than 50% was considered as excessive inconsistency . for all three outcomes , multiple groups from the same study were analyzed independently as well as collapsing multiple groups so that only one es represented each outcome from each study . outliers , considered to be those with standardized residuals yielding an alpha value 0.05 , were deleted from the model in order to examine their influence on the overall findings . in addition , influence analysis was conducted with each result deleted from the model once . were examined using the trim and fill imputation approach ( linear estimator l ) of duval and tweedie . in addition , cumulative meta - analysis , ranked by year , was performed in order to examine the accumulation of results over time . for categorical variables , mixed - effects , anova - like models for meta - analysis were used to compare potential between - group differences ( qb ) . a random - effects model was used to combine studies within each subgroup while a fixed - effect model was used to combine subgroups and yield an overall effect . between - study variance ( ) for all three outcomes , the a priori categorical variables examined included country in which the study was conducted ( usa , other ) , gender , and menopausal status ( pre versus post ) . for exercise dropouts and compliance outcomes , additional moderators examined included type of exercise ( aerobic , strength , or both ) , intensity of exercise ( low , moderate , or high ) , exercise instruction ( supervised , unsupervised , or both ) and setting ( home , facility , or both ) . intensity of training for aerobic exercise was classified as low ( 54% of maximal heart rate or 39% of either maximum oxygen consumption or maximum heart rate reserve ) , moderate ( 55% to 69% of maximal heart rate or 40% to 59% of either maximum oxygen consumption or maximum heart rate reserve ) , or high ( 70% of maximal heart rate or 60% of either maximum oxygen consumption or maximum heart rate reserve ) . for resistance training , intensity was classified as either low ( 49% of maximal voluntary contraction ) , moderate ( 50% to 69% of maximal voluntary contraction ) , or high ( 70% of maximal voluntary contraction [ 18 , 19 ] ) . potential differences in dropout rates between exercise and control groups were also examined using the mixed - effects model . for those potential moderators with more than two groups , paired analyses were conducted if the overall between - group difference ( qb ) was statistically significant . with the exception of country , potential moderators were selected based on previous research showing an association with dropouts and/or compliance [ 2023 ] . the alpha level for statistical significance was set at p 0.05 while alpha values > 0.05 but 0.10 were considered as a trend . simple mixed - effects method of moments metaregression was used to examine the association between exercise and control group dropouts as well as compliance to the exercise protocol . potential predictors included age in years , length of the study in weeks , and the year the study was published . with the exception of publication year , potential moderators were selected based on previous research showing an association with dropouts and/or compliance [ 20 , 24 ] . the alpha level for statistical significance was set at p 0.05 while alpha values > 0.05 but 0.10 were considered as a trend . data were analyzed using comprehensive meta - analysis ( version 2.2 ) , microsoft excel 2007 , and ssc - stat ( version 2.18 ) . after screening 1,055 citations , thirty - six studies representing 3,297 participants ( 1,855 exercise , 1,442 control ) were included [ 2863 ] . a flow diagram for the selection of studies is shown in supplementary file 1 while a general description of the characteristics of each study is shown in supplementary file 2 ( see supplementary material available online at http://dx.doi.org/10.1155/2013/250423 ) . studies were conducted in 11 different countries . these included 14 in the united states [ 2860 , 6063 ] , 5 in canada [ 33 , 34 , 48 , 56 , 61 ] , 4 in australia [ 4446 , 55 ] , 3 in sweden [ 29 , 30 , 35 ] , 2 each in the united kingdom [ 28 , 32 ] , portugal [ 50 , 51 ] or finland [ 39 , 40 ] , and one each in brazil , china , germany , and japan . dropout data were available for 51 exercise groups and 39 control groups while compliance to the exercise intervention was available for 31 groups . the number of groups exceeded the number of studies because some studies included more than one exercise and/or control group . the initial number of participants , per group , ranged from 5 to 123 for exercise dropouts ( x-sd=3623 , median = 30 ) , 6 to 123 for control dropouts ( x-sd=3723 , median = 30 ) , and 14 to 123 for exercise compliance groups ( x-sd=4226 , median = 30 ) . the mean between - group age range for all participants was 23 to 83 years ( x-sd=56.714.7 years , median = 58.7 years ) while initial body weight ranged between 54.1 and 96.2 kg ( x-sd=69.79.7 kg , median = 69 kg ) . thirty - two of 36 studies ( 88.9% ) were limited to women [ 2863 ] , three included both men and women [ 41 , 57 , 58 ] , while one was limited to men . for those studies that reported data and included women , 24 of 36 ( 66.7% ) were limited to postmenopausal women [ 3035 , 37 , 38 , 4145 , 48 , 5058 , 62 ] , 7 to premenopausal women [ 28 , 36 , 39 , 47 , 49 , 59 , 60 ] , and one to perimenopausal women . for the 20 studies that reported data on race / ethnicity [ 33 , 34 , 36 , 38 , 4143 , 46 , 4854 , 5759 , 61 , 62 ] , participants , as described by the original study authors , included whites , asians , hispanics , blacks or african americans and indians . while exact numbers could not be elucidated , the majority of participants appeared to be white . with respect to those studies that reported information on cigarette smoking , 13 reported that none of the subjects smoked [ 34 , 35 , 37 , 3942 , 46 , 47 , 52 , 53 , 58 , 62 ] while 11 reported that some did [ 29 , 30 , 32 , 41 , 43 , 48 , 5052 , 54 , 61 ] . a description of the training program characteristics is shown in supplementary file 2 . as can be seen , the exercise interventions varied . across all intervention groups , length of training ranged from 24 to 104 weeks ( x-sd=54.824.0 , median = 52 ) while frequency ranged from 2 to 7 days per week ( x-sd=3.81.5 , median = 3 ) . for type of exercise , 18 of 51 groups ( 35% ) participated in strength training only , 14 ( 28% ) in aerobic exercise only , and 17 ( 33% ) in both . another two groups ( 4% ) performed either jumping exercises or a combination of resistance training and agility exercises . all of the studies were considered to be at a low risk of bias for sequence generation , an unclear risk for allocation concealment , and a high risk for blinding [ 2863 ] . overall results for exercise and control dropouts as well as compliance to the exercise intervention are shown in table 1 . as can be seen , exercise group dropouts averaged 21% ( 95% ci 17% to 26% ) with a large amount of heterogeneity . a forest plot with the results from each exercise group in each study is shown in figure 1 . results were similar when findings were collapsed so that only one es represented each study ( figure 2 ) . with two outlier studies deleted from the model [ 49 , 60 ] , overall results remained similar ( x-=0.20 , 95% ci , 0.16 to 0.24 , q = 163.4 , p < 0.001 , and i = 70.6% ) while heterogeneity was reduced but still large . with each study deleted from the model once , cumulative meta - analysis , ranked by year , showed that results have remained consistent for approximately a decade ( figure 4 ) . reasons for exercise dropouts for the 21 studies that reported such information [ 2833 , 35 , 3840 , 46 , 48 , 50 , 51 , 53 , 54 , 5759 , 62 , 63 ] included time [ 28 , 33 , 46 , 48 , 54 ] , moving [ 39 , 40 , 54 , 63 ] , loss of interest [ 39 , 40 , 50 ] , injuries which may or may not have been related to the exercise intervention [ 28 , 29 , 32 , 35 , 3840 , 54 ] , personal issues [ 28 , 29 , 50 , 51 , 57 , 58 , 62 ] , medical issues other than injury [ 29 , 32 , 46 , 48 , 50 , 51 , 53 , 57 , 58 , 62 , 63 ] , starting pharmacologic therapy that could affect bmd ( hormone replacement therapy , corticosteroids ) [ 29 , 59 ] , and pregnancy [ 39 , 59 ] . three studies dropped exercise participants because they did not meet their compliance requirements [ 30 , 31 , 47 ] . a forest plot with the control group dropout results from each group in each study is shown in figure 5 . results were similar when findings were collapsed so that only one es represented each study ( figure 6 ) . with two outlier studies deleted from the model [ 49 , 60 ] , results remained similar to group level findings ( x-=0.15 , 95% ci 0.12 to 0.19 , q = 93.9 , p < 0.001 , and i = 61.6% ) . with each study deleted from the model once , the range of control group dropouts was narrow ( 15% to 16% ( figure 7 ) ) . cumulative meta - analysis , ranked by year , showed that results have remained consistent for approximately a decade ( figure 8) . no statistically significant differences were observed in dropout rates between exercise and control groups ( qb = 2.1 , p = 0.14 ) . reasons for control dropouts for the 18 studies that reported such information [ 2830 , 32 , 33 , 35 , 39 , 40 , 46 , 48 , 50 , 51 , 54 , 5759 , 62 , 63 ] included time [ 46 , 48 , 54 ] , moving [ 39 , 54 ] , loss of interest [ 35 , 39 , 40 , 58 ] , personal issues [ 28 , 29 , 50 , 51 , 57 ] , medical issues [ 32 , 33 , 39 , 50 , 51 , 57 , 58 ] , starting pharmacologic therapy that could affect bone ( hormone replacement therapy , corticosteroids ) [ 29 , 33 , 59 ] , pregnancy , started exercising [ 30 , 35 ] , unwilling to serve as a control [ 50 , 51 ] , not available for testing , unsatisfied , unable to cope with trial , and death [ 35 , 40 , 57 ] . a forest plot with compliance results from each group in each study is shown in figure 9 . results were similar when findings were collapsed so that only one es represented each study ( figure 10 ) . with two outlier studies deleted from the model [ 30 , 60 ] , results remained similar to group level findings ( x-=0.76 , 95% ci 0.72 to 0.80 , q = 55.7 , p = 0.001 , and i = 49.8% ) . with each study deleted from the model once , the range for compliance was narrow ( 75% to 76% ) ( figure 11 ) . adjustment for small - study effects ( 9 imputations ) reduced compliance to 72% ( 95% ci = 67% to 77% ) . cumulative meta - analysis , ranked by year , showed that results have remained consistent for approximately a decade ( figure 12 ) . for both exercise and control groups , dropouts were greater for those studies conducted in the usa versus other countries ( exercise = 11% , control = 10% ) , females versus males ( exercise = 19% , control = 12% ) , and premenopausal versus postmenopausal women ( exercise = 22% , control = 18% ) . there was also a statistically significant difference overall when dropout data for exercise groups were partitioned according to intensity of training while a trend was observed for supervision status . two - group comparisons demonstrated that dropouts were approximately 13% greater for high- versus moderate - intensity training ( qb = 7.0 , p = 0.008 ) with no statistically significant differences between high- and low - intensity training ( qb = 2.3 , p = 0.13 ) or moderate- and low - intensity training ( qb = 0.48 , p = 0.49 ) . for supervision status , dropout rates were approximately 17% greater for unsupervised versus a combination of supervised and unsupervised exercise ( qb = 5.0 , p = 0.03 ) . no statistically significant differences were observed between supervised versus a combination of supervised and unsupervised exercise ( qb = 1.4 , p = 0.24 ) or supervised and unsupervised exercise ( qb = 2.4 , p = 0.13 ) . no other statistically significant differences were observed for dropouts and any of the other moderators examined . moderator analysis with respect to exercise compliance demonstrated that adherence to the exercise program was significantly greater ( 13% ) in females versus males . a statistically significant difference was also found for the setting in which exercise took place . two - group comparisons revealed that compliance was significantly greater ( 21% ) in facility versus a combination of facility- and home - based exercise ( qb = 4.8 , p = 0.03 ) as well as 28% greater for home versus a combination of facility- and home - based exercise ( qb = 7.2 , p = 0.007 ) . no statistically significant differences were found when data were partitioned according to facility- versus home - based exercise ( qb = 1.8 , p = 0.18 ) . no other statistically significant differences were observed for compliance and any of the other moderators examined . simple metaregression results for exercise and control group dropouts as well as compliance to the exercise protocol are shown table 3 . for both exercise and control groups , dropout rates were significantly lower with increasing age ( r = 0.32 for exercise and 0.25 for control groups ) . there was also a statistically significant association between greater dropout rates in the control groups and longer interventions ( r = 0.11 ) . for compliance , for all three outcomes , no other statistically significant associations were observed for any of the other potential predictors examined . the primary purpose of this aggregate data meta - analysis was to examine dropouts and compliance in exercise interventions targeting bmd in adult humans . collectively , 21% of exercise group participants and 16% of control group participants dropped out of the study while another 24% did not fully comply with the exercise intervention . , a meta - analysis of 24 large observational studies found that adherence to drug therapies for osteoporosis ranged from 40% to 70% . in addition , one must consider the potential side effects and costs associated with pharmacologic interventions . for both exercise and control groups , dropout rates were significantly greater for studies conducted in the united states versus other countries . while purely speculative , one of the possible reasons for the difference may be related to potentially stricter institutional review board rules in the usa versus other countries , thereby making it easier to withdraw from the study . alternatively , us participants may be less motivated to maintain a regular exercise program . greater dropout rates were observed for females versus males in both exercise and control groups while greater compliance was observed for females . these findings suggest that those females who remain in an exercise program are more likely than males to comply with the prescribed program . a recent systematic review by pavey et al . found that women were more likely than men to begin an exercise referral scheme but less likely to adhere to it . the greater exercise and control group dropout rates found for pre- versus postmenopausal women suggests that older women are less likely to drop out of an exercise program . this is supported by the inverse association that was found between age and dropout rates for both exercise and controls . this finding is further supported by a recent systematic review that found that older people were more likely to begin and adhere to an exercise referral scheme . the lower dropout rate and higher compliance rate for older versus younger participants may reflect a higher level of interest on their part and/or a greater amount of time to devote to exercise . while exercise group dropout rates were progressively greater as the intensity of exercise increased , the only statistically significant difference was between high- and moderate - intensity training . however , the lack of a statistically significant difference between high- and low - intensity training ( 20% greater for high- versus low - intensity training ) may have been the result of the small sample size available ( n = 3 ) for low - intensity training . in contrast to the exercise and control group findings , no statistically significant differences were found between compliance and intensity of training . this is in contrast to a recent study that found that the compliance was greater with moderate versus high - intensity aerobic exercise . one of the potential reasons for this difference may be the fact the both aerobic and resistance training interventions were included in the current investigation while the previous study by perri et al . was limited to one aerobic activity ( walking ) . another possible reason for the lack of a statistically significant difference for compliance in the current meta - analysis may have to do with the fact that three included studies dropped exercise participants because they did not meet the study 's compliance requirements [ 30 , 31 , 47 ] . it is generally accepted that greater benefits are usually obtained from higher versus lower intensity training programs . however , this needs to be balanced with participant dropout and lower compliance as well as the possibility for an increased risk of injury . thus , while one should probably not dissuade a participant from higher intensity training , greater adherence may be achieved with moderate ( e.g. , walking briskly ) versus higher ( e.g. , running ) exercise . while there was a trend ( p = 0.08 ) for an overall difference in dropout rates between supervised , unsupervised , or combined supervised and unsupervised exercise , subgroup analyses revealed greater dropout rates in unsupervised versus a combination of supervised and unsupervised exercise . this is in contrast to a recent systematic review that suggested that supervised exercise may enhance adherence . while future research in this area appears warranted , it is generally believed that the greater the amount of attention that participants receive , the lower the dropout rate and the greater the compliance . study length was associated with greater dropout rates in the control groups and poorer compliance in the exercise groups . while no statistically significant association was found for exercise dropouts the greater dropout rates in the control groups over time may be the result of participants losing interest because of the lack of attention they receive during the study period . it may also be the result of their desire to be assigned to the exercise intervention when they enrolled . the findings of the current meta - analysis support a previous narrative review that found that exercise participation declines over time . while no statistically significant difference was found between types of exercise ( aerobic , strength training , or both ) , exercise dropouts , and compliance , a recent systematic review with meta - analysis found that resistance exercise predicted higher attendance rates than aerobic exercise in sedentary older adults . the same review also found higher completion rates with facility- versus home - based exercise while the current meta - analysis found greater compliance for facility- or home - based exercise versus both but no difference between home- or facility - based exercise ( p = 0.18 ) . based on these findings , it would seem appropriate to suggest that future research in this area is warranted . for example , the results for dropout and compliance rates may be helpful with respect to sample size estimation for researchers planning future randomized controlled trials . factors that appear to be important when attempting to minimize dropouts and maximize compliance include ( 1 ) gender , ( 2 ) age , ( 3 ) exercise intensity ( low , moderate , or high ) , ( 4 ) exercise supervision ( supervised , unsupervised , or both ) , ( 5 ) setting ( home , facility , or both ) , and ( 6 ) study length . in addition , researchers can do a better job in reporting the reasons for dropouts . this includes reporting information separately for each group ( exercise and control ) , including the number of participants , per group , associated with the reason for dropping out . given the lack of information provided by some studies for compliance , future studies should also make sure to report this information . furthermore , future studies should report the method used to conceal the allocation sequence in sufficient detail to determine whether assignments could have been anticipated prior to or during enrollment . it is also important to note that while blinding was considered to be at a high risk for bias across all studies , it is not possible to blind participants in exercise intervention studies . the results of the current study suggest that there are several factors that should be considered for practitioners , defined here as those individuals responsible for developing , implementing , and evaluating exercise programs . these include ( 1 ) gender , ( 2 ) age , ( 3 ) exercise intensity ( low , moderate , or high ) , ( 4 ) exercise supervision ( supervised , unsupervised , or both ) , and ( 5 ) setting ( home , facility , both ) . while maximizing long - term participation is important , this has to be balanced with available resources . while the results of the current meta - analysis provide some interesting findings , they should be interpreted with respect to the following potential limitations . first , because of missing data for different variables from different studies as well as small sample sizes within selected subgroups , multiple meta - regression analysis was not performed . as a result , potential confounding factors were not controlled for . second , one or more of the statistically significant findings may have been nothing more than the play of chance given the large number of statistical tests conducted . however , an a priori decision was made to not adjust alpha values because such adjustments tend to be overly conservative . furthermore , the investigative team did not want to miss any potentially important findings that might be worthy of further investigation . third , while the current study included a number of potential moderators , many other potential determinants of exercise adherence exist but are not typically reported in randomized controlled trials . broadly , these include such things as personal ( self - efficacy , enjoyment of activity , etc . ) and environmental ( spousal support , disruptions in routine , etc . ) characteristics . finally , the results of the current meta - analysis may not be generalizable beyond the participants and interventions that were included . the current study addresses a recent recommendation for additional research on the determinants of exercise . it provides important evidence regarding dropouts and compliance , including factors associated with dropouts and compliance , for exercise interventions targeting bmd in adult humans .

background . dropouts and compliance to exercise interventions targeting bone mineral density ( bmd ) in adults are not well established . the purpose of this study was to address that gap . methods . meta - analysis of randomized controlled exercise intervention trials in adults 18 years of age . the primary outcomes were dropouts in the exercise and control groups as well as compliance to the exercise interventions . a random - effects model was used to pool results . moderator analyses were conducted using mixed - effects anova - like models and metaregression . statistical significance was set at p 0.05 . results . thirty - six studies representing 3,297 participants ( 1,855 exercise , 1,442 control ) were included . dropout rates in the exercise and control groups averaged 20.9% ( 95% ci 16.7%25.9% ) and 15.9% ( 11.8%21.1% ) while compliance to exercise was 76.3% ( 71.7%80.3% ) . for both exercise and control groups , greater dropout rates were associated with studies conducted in the usa versus other countries , females versus males , premenopausal versus postmenopausal women , younger versus older participants , longer studies ( controls only ) , and high- versus moderate - intensity training ( exercisers only ) . greater compliance to exercise was associated with being female , home- or facility - based exercise versus both , and shorter studies . conclusion . these findings provide important information for researchers and practitioners with respect to exercise programs targeting bmd in adults .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions

PMC4648225

in recent years , a significant increase in the number of scientific health publications has been registered . there is no science without being published , as it corresponds to the permanent record of our research , reputation and immortality ; ( b ) the author s motivation to publish due to their need to obtain funding and further their career ; ( c ) the perception that to publish is the individual or team effort to ensure the wide sharing of results ; on the other hand , to not publish may suggest that the author is not committed to sharing knowledge and , in some cases , wishes to avoid scientific discussion with peers . perhaps in the past it was more difficult to publish than it is at the present . database access to publications was scarce or nonexistent , and the cycle of publication was time consuming without an online platform . nevertheless , the competition to publish was not as aggressive , the impact factor and the h index were not a concern , and scientists were not constantly scrutinized according to their publication records / numbers . nowadays , these aspects have become new worries , increased by new ethical and conflict of interest issues . thus , to publish is both almost compulsory ( a question of survival ) and simultaneously a very hard task ; we are in the era of hunting the article , which in some cases may promote fraud and corruption . how could we explain the 1.7 to 1.8 million articles in 2013 in journals with peer - review ( a supposed certificate of quality ) , or approximately one article every 18 seconds ? moreover , and in spite of significant innovation , some studies suggest that the average peer - review takes approximately 10.9 and 6.5 hours for young versus experienced reviewers , respectively . in addition , according to mabe the number of new peer reviewed journals and articles published annually has been growing at a very steady rate of about 33.5% per year for over three centuries , but in the last few years this increase has become more pronounced . publishing is also a business and some authors even suggest that it is becoming pathological , with psychological and legal implications . the international association of scientific , technical and medical ( stm ) publishers that holds 66% of the publication market and each year publishes nearly two - thirds of all journal articles , handled $ 9.4 billion in 2011 ( up from $ 8 billion comparatively in 2008 ) solely in scientific journals , and employs about 110,000 people . although the united states of america ( usa ) continues to dominate the global production of research papers , significant growth has also been registered for china and east asia . this commentary aims to highlight some relevant aspects of scientific publications that all health care providers and researchers , as well as medical students , should better understand in order to avoid publication misconduct . in traditional journals , articles are usually behind a pay - wall , meaning that readers must have a subscription or pay a fee to read content . in opposition to this traditional method , the number of open access ( oa ) journals have been increasing in the last few years . oa journal models comprise two main approaches : this model of oa publishing has been increasing in popularity and represents direct publication in oa journals , with the majority requiring payment of the cost of publication . nevertheless , some are sponsored , which means that the author does not pay or some institutions may have an agreement with publishers ( i.e. the oa membership ) . a hybrid or optional variant exists ( i.e. only part of the article is immediately oa and , if wanted , the author may pay for the full article to be oa ) ; in this case , some traditional journals , after an embargo ( necessary to recover the investment by publishers ) , allow the publication of some articles in free repositories ( e.g. medline , psydok ) . for free access after the embargo there are around 10,128 fully oa journals listed on the directory of open access journals . this publication model is important for future citation and therefore to maximize the impact of research . the clients of the publishers are the authors and not the readers , but the exaggeration in the number of journals and articles may even disquiet the best proponents of the oa model . for example , in 2013 plos one published approximately 31,500 articles ( a staggering increase from 138 articles in 2006 ) , meaning almost one in every 60 pubmed articles if from plos one . structure is widely accepted as one of the most trusted oa journals , practicing a very broad scope and a rapid non - selective peer review based on soundness not significance ( i.e. selecting papers on the basis that science is soundly conducted rather than more subjective criteria of impact , significance or relevance to a particular community ) . currently , brazil is the country with the most oa journals after the usa ; more than one thousand . that abundance was recently described as a plague of brazilian science : articles of second class . in our opinion one of the best papers alerting the community to this problem the author investigates the peer - review process of the fee - charging oa journals . between january and august 2013 , bohannon submitted obvious false articles to 304 scientific journals . in the author s opinion the article should have been immediately rejected by the editors and reviewers , but 60% of journals accepted it . the article was based on multiple combinations of the x molecule of species y lichens that inhibits the growth of cancer cells z . for each article , the study also showed a map of the geographical location of the editors and publishers and their bank accounts . the author concluded that any reviewer with basic knowledge of chemistry and with skills to analyze an elementary graph should have immediately detected the article s faults . he concluded that it was easy to jump directly from the test tube to the clinic . subsequently , hvistendahl reported that in china , a black market has been developed by some agencies to allow authors to write articles without a need to perform experimental work . the prices depend on whether the person paying wishes to be listed as the primary writer , or as merely a co - author , or even as just one of the team members . authors suggested that this black market may have contributed to china s rapid growth in the number of science citation index ( sci ) articles , with china s contribution now the second largest , after the usa . curiously , a running joke in china offered another meaning for sci : stupid chinese idea . furthermore , researchers are bothered with several invitations to review or submit articles that are not within their main area of research , and the same invitations are distributed to many email accounts . perhaps also sharing the same annoyance at this state of affairs , jeffrey beall in august 2012 published the first edition of the criteria for determining predatory open - access publishers . these criteria were the start point to generate a list of potential , possible , or probable predatory oa scholarly publishers . in fact , they are a very positive way to share scientific knowledge for all researchers , and many possess great credibility . the question now is what journals we should choose and how to assess their credibility . to help to solve this problem , it is now possible for readers to perform a continuous re - reviewing of published articles as it occurs for f1000research . through the readers opinions this model of oa publishing has been increasing in popularity and represents direct publication in oa journals , with the majority requiring payment of the cost of publication . nevertheless , some are sponsored , which means that the author does not pay or some institutions may have an agreement with publishers ( i.e. the oa membership ) . a hybrid or optional variant exists ( i.e. only part of the article is immediately oa and , if wanted , the author may pay for the full article to be oa ) ; in this case , some traditional journals , after an embargo ( necessary to recover the investment by publishers ) , allow the publication of some articles in free repositories ( e.g. medline , psydok ) . for free access after the embargo there are around 10,128 fully oa journals listed on the directory of open access journals . this publication model is important for future citation and therefore to maximize the impact of research . the clients of the publishers are the authors and not the readers , but the exaggeration in the number of journals and articles may even disquiet the best proponents of the oa model . for example , in 2013 plos one published approximately 31,500 articles ( a staggering increase from 138 articles in 2006 ) , meaning almost one in every 60 pubmed articles if from plos one . structure is widely accepted as one of the most trusted oa journals , practicing a very broad scope and a rapid non - selective peer review based on soundness not significance ( i.e. selecting papers on the basis that science is soundly conducted rather than more subjective criteria of impact , significance or relevance to a particular community ) . currently , brazil is the country with the most oa journals after the usa ; more than one thousand . that abundance was recently described as a plague of brazilian science : articles of second class . in our opinion one of the best papers alerting the community to this problem the author investigates the peer - review process of the fee - charging oa journals . between january and august 2013 , s opinion the article should have been immediately rejected by the editors and reviewers , but 60% of journals accepted it . the article was based on multiple combinations of the x molecule of species y lichens that inhibits the growth of cancer cells z . for each article , the study also showed a map of the geographical location of the editors and publishers and their bank accounts . the author concluded that any reviewer with basic knowledge of chemistry and with skills to analyze an elementary graph should have immediately detected the article s faults . he concluded that it was easy to jump directly from the test tube to the clinic . subsequently , hvistendahl reported that in china , a black market has been developed by some agencies to allow authors to write articles without a need to perform experimental work . the prices depend on whether the person paying wishes to be listed as the primary writer , or as merely a co - author , or even as just one of the team members . authors suggested that this black market may have contributed to china s rapid growth in the number of science citation index ( sci ) articles , with china s contribution now the second largest , after the usa . curiously , a running joke in china offered another meaning for sci : stupid chinese idea . furthermore , researchers are bothered with several invitations to review or submit articles that are not within their main area of research , and the same invitations are distributed to many email accounts . perhaps also sharing the same annoyance at this state of affairs , jeffrey beall in august 2012 published the first edition of the criteria for determining predatory open - access publishers . these criteria were the start point to generate a list of potential , possible , or probable predatory oa scholarly publishers . in fact , they are a very positive way to share scientific knowledge for all researchers , and many possess great credibility . the question now is what journals we should choose and how to assess their credibility . to help to solve this problem , it is now possible for readers to perform a continuous re - reviewing of published articles as it occurs for f1000research . through the readers opinions peer review is a methodological evaluation on the soundness of the topic , originality , methodology , results and conclusions highlighted by the author and the authorities cited . although it can not generally assure that the data is truthful or not , peer review unquestionably increases the quality of most manuscripts nevertheless , this procedure is sometimes slow , expensive , profligate , subjective , prone to bias , poor at detecting gross defects , and almost useless in detecting fraud . typically pre- ( e.g. double and single - blind , and open ) or post - publication peer review is practiced . double - blind peer review is more common in the humanities and social sciences than in the exact sciences . although the identity of the reviewers is not disclosed and vice - versa ( which removes the potential influence of the involved countries , institutions or eventual personal conflicts ) , the process sometimes fails , because the method , style of writing , acknowledgments and abuse of self - citations can suggest the source . in single - blind peer review the author is unaware of the identity of the reviewers ; it is the most common process , especially in life sciences and is useful for the reviewer in order to consult previous authors works . by the same reasons listed for the double - blind peer - review although less common than the previous peer reviews , the open peer review process is increasing : nevertheless , it results in less acceptances to review since reviewers are afraid of being identified if commenting negatively on the article . nevertheless , it is expected that reviewers produce better revisions and avoid offensive or rude words . the name of the reviewers and comments may be published alongside the article as it occurs in the british medical journal . once the pre - publication peer review process is completed , the decision to publish is made by the journal editor on the advice of the reviewers . the editor of a journal is usually an independent , leading expert in his / her field appointed and sometimes financially supported by the publisher . an apparent misuse of editorial privileges was practiced by the editor of chaos , solitons and fractals ( a theoretical - physics journal ) , who was criticized by using its pages to publish numerous articles written by himself ( e.g. 36 papers in the december 2008 issue ) . finally , in post - publication peer review , the quality is assessed by the sapient of crowds and readers can also judge the quality of the review process . this peer review is outside the editor s monopoly and journals usually also provide a discussion forum about the article in order for researchers to comment on and read other s comments . similar to the british medical journal , referee reports and names are published alongside the article , together with the authors ' responses to raised points . it is believed that both invited and un - invited ( i.e. , commenting ) post publication peer review helps to increase the quality of the final publication and it is certainly more transparent . f1000research follows this model and advocates that time is not lost in reviews , since each article is only subjected to editorial verification and within approximately one week is published online already formatted and can be read and cited ( http://f1000research.com/about ) . in 1960 , the institute for scientific information ( isi ) was founded by eugene garfield and later he proposed the impact factor ( if ) as a tool for journal evaluation by librarians to help them with journal purchasing decisions . the isi was purchased by thomson scientific & healthcare in 1992 becoming the thomson isi . if it is not a perfect tool to measure the prestige of the journal , but there is no better . falagas and colleagues performed a very interesting discussion on metrics for scientific journals as well as for researchers . some biases of the if include : ( a ) it is not statistically representative of individual articles ; ( b ) review articles ( often highly cited ) greatly skew the results ; ( c ) extensive articles always have many citations and yield a high if ; ( d ) it includes self - citations ; ( e ) books do not count for citations ; ( f ) databases primarily index articles in english ( but the if also exists for non - english journals ) and are dominated by usa publications ; ( g ) it is dynamic since it depends on fluctuations of research in a given area ; ( h ) paid access of some journals ; ( i ) journals with tight scope tend to have low if ; ( j ) it does not take into account the subjects variability ( e.g. , immunology and cancer are usually highly cited ) ; ( k ) editors aware of the importance of the if tend to accept articles that may be highly cited and to reduce the number of articles accepted ; ( l ) the absence of an if in a given journal can result in low submissions ; ( m ) it is only applied to isi journals ; ( n ) one highly cited article can boost the if in a given journal ( e.g. , the impressive if of 49.9 for acta crystallographica section a : foundations of crystallography ; the primary cause of this high impact factor was a single feature article by sheldrick ) . nowadays , there is rising interest in research and publication ethics . proof of that is the increased importance of organizations such as the committee on publication ethics ( cope ) and the development of software to detect plagiarism . although , the number of journal article retractions has grown in the last decade , it is the general consensus that this may be the result of increased awareness rather than misconduct . andrew jeremy wakefield , a former british surgeon and researcher , published a fraudulent study in 1998 claiming that there was an association between the administration of the measles , mumps and rubella vaccine , and the development of autism and crohn s disease 20 , 21 . the german physicist jan hendrik schn , starred a scandal related to semiconductors that triggered a series of retractions , six of them out of science . hwang woo - suk ( i.e. the pride of south korea ) was sentenced to two years in prison with suspended sentence after distorting the results published in two science articles related to cloning of human embryonic stem cells 23 , 24 . more recently in january 2014 , haruko obokata , a young researcher in japan published in nature , showed stem cells can now be made quickly just by dipping blood cells into acid 25 , 26 . on june 2014 , obokata agreed to retract both the papers and 2 months later obokata s mentor and co - author , yoshiki sasai , committed suicide by hanging . although investigation cleared him of misconduct , he was not free of critiques for inadequate supervision of haruko obokata . in 2005 , the researchers david mazires and eddie kohler designed an anecdote manuscript , to send in response to unsolicited congress invitations . later in 2014 , peter vamplew , an associate professor at the federation university australia school of engineering and information technology , after receiving a spam email from the international journal of advanced computer technology ( classified as predatory oa on beale s list ) , forwarded mazires and kohler s old paper as a response . the manuscript was not actually published since vamplew declined to pay the article processing charge . acceptance of a paper consisting entirely of 863 repetitions of get me off your fucking mailing list has led commenters to question whether the enterprise is more interested in collecting publication fees than in contributing first - rate peer reviewed articles to the advancement of computer science . some epic / record examples of fraud were practiced by joachim boldt and yoshitaka fujii . joachim boldt is a german anesthesiologist who was dismissed of his professorship and is under criminal investigation for having allegedly faked of up to 90 research studies . yoshitaka fujii is a japanese researcher in anesthesiology , who in 2012 was found to have fabricated data in at least 172 scientific papers over the past 19 years , setting what is believed to be a record for the number of papers by a single author requiring retractions 29 , 30 . many of the listed co - authors did not know they were authors and their signatures had been forged in the copyright transfer . scientists do not have schedules and need to fight for financial support ; their careers grow exponentially when they publish ( especially in high if journals ) and their job security frequently depends on the number of publications . but first of all , scientists are human beings , with families , needs and emotions . therefore , the motivation to be successful and remain employed may increase the risk of involvement in scientific fraud or even corruption . is the impact of fraud in areas that have significant impact on the health , safety and welfare of the world s population , as are the cases of life and health sciences . moreover , scientific research slows since it is necessary to spend more time confirming published results . taradi and colleagues show that over 90% of the medical students of croatia admitted to engaging in education dishonesty and over 78% engaging in academic misconduct . indeed , fraud can help the scientist rise , but also fall rapidly . it is also relevant to make peer review more transparent to produce publications that are more genuine and free from bias . the increase in publications , research studies split into multiple publications ( rather than single , longer articles ) , the proliferation of journals , the ways in which academic promotion fosters this proliferation of publications , and the ways in which these changes can encourage bad or even fraudulent science are important topics that will certainly dictate the future of life and health sciences research nevertheless , we will face problematic times if financial issues superimpose the ethics in publishing . this crisis may be an opportunity and challenge to reflect on these topics .

in recent years , there has been a significant increase in the number of scientific publications it is the era of hunting the article . this commentary discusses the drawbacks of the pressure to publish that certainly contribute to the dark side of science . in fact , health science career progression greatly relies on the number of scientific publications a researcher has , and in many cases these may be more valorized than the health services provided . of course , scientific publications help to develop the skills of health care professionals , but as einstein highlighted not everything that counts can be counted , and not everything that can be counted counts .

Introduction Concerns in selecting scientific journals 1. OA publishing or the Gold route 2. Delayed free access and self-archiving, or the Green route Constraints of the peer review cycle Limitations of the impact factor Fraud in life sciences Concluding remarks

PMC2939353

infections of medical prosthetic devices in trauma and orthopaedic surgery are difficult to treat with conventional therapies like antibiotics and surgery , and can lead to severe consequences , such as removal of the implant with functional loss of the affected limb [ 18 , 26 ] . the most frequently isolated bacteria causing these implant- and patient - related infections are s. aureus and the coagulase - negative s. epidermidis [ 1 , 11 ] . less common are pseudomonas aeruginosa , klebsiella oxytoca , enterococcus faecalis , and enterobacter cloacae . in prosthesis - associated infections , the causative bacteria form biofilms composed mainly of a polysaccharide matrix . the most important difference between bacteria in biofilms and planktonic microorganisms that can explain the alteration in antibiotic resistance is the metabolic state of biofilm bacteria . the genotype and phenotype of bacteria in biofilms and the protein expression are altered and the oxygen concentration in biofilms is limited . these changes result in metabolic quiescence , which could be overcome by providing alternative electron acceptors of fermentable substrates [ 3 , 24 ] . current antimicrobial therapies frequently can not adequately treat biofilm - associated infections [ 8 , 9 , 24 , 26 ] and therefore it is important to develop new treatment approaches to reduce biofilm formation ( bf ) on biomaterials . the most important development in the treatment of infectious diseases during the last century was the discovery of penicillin by fleming in 1928 . at approximately the same time , maggot debridement therapy ( mdt ) was introduced by william s. baer , an orthopaedic surgeon who worked at johns hopkins hospital in baltimore , maryland . although baer showed the beneficial effects of maggots of the lucilia sericata , mdt essentially disappeared after the discovery of penicillin , which could control numerous types of severe infections . because of increasing antibiotic resistance , larval therapy was reintroduced in the 1980s as a treatment for severely infected wounds maggots now are used successfully in various patient clinics [ 13 , 25 ] . in 2004 , mdt was approved by the us food and drug administration ( 510[k ] # 33391 ) . in our clinical practice , we use maggots captured in small nylon bags , consisting of a 2-mm thin foam layer of polyvinyl alcohol ( biobag ; biomonde gmbh , barsbttel , germany ) . we have observed healing of wound infections suspect for bf using maggots in such bags . in an earlier pilot study however , the current research investigated , for the first time , the influence of a range of concentrations of es on mature 7-day - old biofilms formed by new , commonly isolated bacterial species on different biomaterials . if maggot es reduce bf of bacteria that frequently are isolated from biofilm - associated infections , es could provide us with a new treatment possibility . therefore , we asked ( 1 ) whether five bacterial species ( s. aureus , s. epidermidis , k. oxytoca , e. faecalis , and e. cloacae ) , were able to form biofilms on polyethylene ( pe ) , titanium ( ti ) , and/or surgical stainless steel ( sss ) , and if so , whether ( 2 ) the quantity of bf differed among the bacterial species , ( 3 ) the quantity of bf depended on the biomaterial surface , and ( 4 ) the quantity of biofilms of the biofilm - forming bacteria differed among 3 , 5 , 7 , or 9 days of incubation time . finally , we hypothesized ( 5 ) sterile maggot es could decrease the growth of the bacterial biofilms on the commonly used biomaterials sss , ti , and pe . first , we investigated bf of five different , clinical isolated species on comb - like models of pe , ti , and sss suspending in a 96-well microtiter plate with nutrient medium and bacteria ( fig . 1 ) . two of the five tested bacterial species formed visible biofilms that could be quantified and these bacteria were used to investigate the second to fifth research questions . the dependent variable in the second to fifth research questions was the quantity of formed biofilm ; bf was compared among the two biofilm - forming species , among the different biomaterials sss , ti , and pe and between various incubation times on days 5 , 7 , and 9 . bf = biofilm formation ; pe = polyethylene ; ti = titanium ; sss = surgical stainless steel ; es = excretions and secretions . bf = biofilm formation ; pe = polyethylene ; ti = titanium ; sss = surgical stainless steel ; es = excretions and secretions . the custom - made , sterile comb - like models of regularly available orthopaedic implant materials , consisting of eight prongs were produced according to our design . these models of sss , ti , and pe ( litos gmbh , hamburg , germany ) were made to fit in a 96-well microtiter plate . s. aureus , s. epidermidis , k. oxytoca , e. faecalis , and e. cloacae all were isolated from infected prosthetic devices of patients in our clinic . briefly , s. aureus and s. epidermidis were grown overnight in tryptic soy broth ( tsb ) medium ( becton , dickinson & co , franklin lakes , nj ) and k. oxytoca , e. faecalis , and e. cloacae in brain heart infusion ( bhi ) medium ( becton , dickinson & co ) at 37c . a stationary phase culture was made in either tsb or bhi to a density of mcfarland 0.5 corresponding with 1.5 10 colony - forming units ( cfus ) per l . the inoculum was controlled by inoculating nutrient agar plates ( biotrading benelux bv , mijdrecht , the netherlands ) with 10-l volumes of serial 1:10 dilutions of the bacterial suspension and counting the formed cfus after incubation overnight at 37c . the bacterial suspension was diluted to a final inoculum of 2.5 10 bacteria per ml and a volume of 100 l of this suspension was pipetted into 24 wells of a sterile 96-well flat - bottomed microtiter plate ( greiner bio - one bv , monroe , nc ) . one comb of each biomaterial was placed in eight of the wells and four of these microtiter plates with combs were incubated for 3 , 5 , 7 , or 9 days at 37c to form biofilms on the devices . control combs were suspended in nutrient medium . after the incubation time , each comb was washed under slow - running distilled water at room temperature for 30 seconds to remove planktonic cells . all comb models were stained for 15 minutes with 1% crystal violet , based on the methods of pitts et al . and stepanovic et al . . each comb was suspended in eight wells of a new microtiter plate filled with 270 l ethanol absolute to absorb the crystal violet . the plates were left for 48 hours at room temperature to allow the stained biofilm bacteria to elute with ethanol . all stained biofilm bacteria are soluble in ethanol , however the biofilm on pe could not be eluted entirely in ethanol ( maximum time tested was 96 hours ) , but we standardized the time of elution to 48 hours to guarantee reliable interpretation of the results . after gentle shaking of the microtiter plate , a volume of 150 l of the ethanol and crystal violet solution was pipetted into a new plate and the optical density ( od ) of this solution was measured at a wavelength of 595 nm and is representative for the quantity of biofilm formation . biofilms of each bacterial species were formed at least in octuplicate , so that one comb was tested with each of the eight prongs suspended in a separate well with nutrient medium and bacterial suspension . sterile maggot es of instar-3 larvae ( biomonde gmbh ) were collected as described previously . briefly , the maggots were incubated in sterile tubes for 1 hour at 35c in darkness . then , es were removed by pipette , divided in aliquots , and stored at 80c . one es pool was collected from at least 1000 maggots , which resulted in approximately 1200 l es per hour . the protein concentration was determined using the pierce bicinchoninic acid protein assay kit ( pierce biotechnology , rockford , il ) . absolute od values of the control bf after 7 days without es are reported in results . to compute absolute and relative od , we first subtracted the material - specific background absorbance . the measured od values of the biofilm experiments were converted to percentages using the following formula:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left [ { { \text{od}}\,\left ( { \text{biofilm } } \right ) \times 100\% } \right]/{\text{od}}\,\left ( { \text{control } } \right ) $ $ \end{document}where od ( biofilm ) refers to either bf measured on day 3 , 5 or 9 ( without es ) or bf measured after the addition of increasing concentrations of es and where od ( control ) is the bf after 7 days on the negative control prongs . in all experiments , biofilms were allowed to form on 13 combs of each of the materials for 7 days , two other combs were suspended in nutrient medium without bacteria as negative controls and represented the material - specific background absorbance . two other combs were suspended in a new microtiter plate with medium and incubated another 24 hours to allow additional growth of the biofilm ( the 8-day - old biofilms ) ; nine combs were suspended in a logarithmic range of concentrations of es , diluted in nutrient medium : 0.31 , 0.93 , 2.78 , 8.33 , 25 , and 75 g es per well . all wells were incubated another 24 hours at 37c and then we quantified the biofilm as described previously . biofilm inhibition ( or prevention ) by es was defined as bf that was lower than 8-day - old biofilms which grew further after day 7 . bf that was lower than 7-day - old bf after incubation in es was defined as biofilm breakdown . the first research question was not analyzed statistically because the results were obtained by observation only . for the second and the last research questions , student s t test for independent groups was used . the means of the 7-day - old biofilms of the biofilm - forming bacterial species were compared to analyze whether bf depended on the bacterial species . furthermore , the mean bf with es ( per concentration ) and the mean of the 8-day - old biofilm without es were compared . for the third research question , one - way anova was used and followed by the least significance difference post hoc test . all analyses met the assumption of homogeneity of variances ( levene s statistic : p > 0.05 ) . to analyze whether the bf depended on the material surface , the mean quantity of a 7-day - old biofilm was compared among the materials for each bacterium . the mean quantities of biofilms formed on days 5 , 7 , and 9 for each bacterium were analyzed using repeated - measures anova followed by the bonferroni correction to investigate whether the bf changed among these 3 days . all analyses met the assumption of sphericity ( mauchly s test : p > 0.05 ) . all statistical analyses were performed using spss for windows , version 14.0 ( spss inc , chicago , il ) . all tested bacterial species were able to form visible biofilms on pe , sss , and ti , however biofilms of k. oxytoca , e. faecalis , and e. cloacae could barely be observed , in contrast to the clearly visible biofilms of s. aureus and s. epidermidis . quantities of biofilm formed by k. oxytoca , e. faecalis , and e. cloacae were insufficient to be measured . therefore , we focused on bf by s. aureus and s. epidermidis and continued the experiments with these two bacterial species . no differences in quantity were seen on pe ( p = 0.582 ) , sss ( p = 0.051 ) , or ti ( p = 0.178 ) ( fig . 2).fig . 2a bthe ods reflect the quantities of 7-day - old biofilms produced by ( a ) s. aureus and ( b ) s. epidermidis on pe , sss , and ti and answer the second and the third research questions , respectively , whether the quantity of bf differed among s. aureus and s. epidermidis and whether bf depended on the biomaterial surface . both bacteria formed equal quantities of bf and showed pe had the highest bf followed by sss and ti with the lowest quantity of biofilm . * for these comparisons the ods reflect the quantities of 7-day - old biofilms produced by ( a ) s. aureus and ( b ) s. epidermidis on pe , sss , and ti and answer the second and the third research questions , respectively , whether the quantity of bf differed among s. aureus and s. epidermidis and whether bf depended on the biomaterial surface . both bacteria formed equal quantities of bf and showed pe had the highest bf followed by sss and ti with the lowest quantity of biofilm . bf by s. aureus was greater on pe ( od595 0.315 ) than on sss ( od595 0.190 ) ( p = 0.024 ) and ti ( od595 0.0386 ) ( p < 0.001 ) ( fig . 2a ; table 1 ) and the same trend was seen for bf by s. epidermidis ( pe : od595 0.248 , sss : od595 0.104 [ p < 0.001 ] ; ti : od595 0.0578 [ p < 0.001 ] ) ( fig . 2b ; table 2).table 1optical density ( 595 nm ) for a 7-day - old biofilm by s. aureusmaterial7-day - old biofilmbackground absorbancepolyethylene0.3150.167surgical stainless steel0.1900.101titanium0.03860.0801table 2optical density ( 595 nm ) for a 7-day - old biofilm by s. epidermidismaterial7-day - old biofilmbackground absorbancepolyethylene0.2480.162surgical stainless steel0.1040.0800titanium0.05780.0740 optical density ( 595 nm ) for a 7-day - old biofilm by s. aureus optical density ( 595 nm ) for a 7-day - old biofilm by s. epidermidis s. aureus and s. epidermidis were observed for bf during 3 , 5 , 7 , and 9 days . on day 3 , biofilms of both bacteria occurred in insufficient quantity to be measured . on ti , pe , and sss , most biofilm was formed by s. aureus on day 7 and day 9 ( day 7 versus day 9 [ all p values 0.198 ] ) ( fig . 3a ) . the amount of biofilm formed by s. epidermidis on pe was equal at all times of incubation ( day 5 versus day 7 [ p = 1.000 ] , day 5 versus day 9 [ p = 0.074 ] ) ( fig . the greatest quantity of biofilms was formed by s. epidermidis on sss and ti ( day 7 versus day 9 [ all p values 0.141 ] ) ( fig . 3a bthe graphs show the amount of bf after various incubation times and answer the fourth research question , whether quantity of biofilms differed among 3 , 5 , 7 , or 9 days . ( a ) biofilms by s. aureus kept on growing until day 9 on pe , ti , and sss . ( b ) the largest amount of biofilm by s. epidermidis was formed within 7 to 9 days on sss and ti . * for these comparisons all p values < 0.001 ; all other comparisons were similar . the graphs show the amount of bf after various incubation times and answer the fourth research question , whether quantity of biofilms differed among 3 , 5 , 7 , or 9 days . ( a ) biofilms by s. aureus kept on growing until day 9 on pe , ti , and sss . ( b ) the largest amount of biofilm by s. epidermidis was formed within 7 to 9 days on sss and ti . the addition of es to 7-day - old biofilms of s. aureus and s. epidermidis reduced these biofilms compared with biofilms without addition of es on all tested biomaterials ( figs . 4 , 5 ) . the biofilm of s. aureus on sss and pe decreased for all concentrations of es to minimums of 40.5% on sss for 0.31 g es per well ( p < 0.001 ) and 39.2% on pe for 0.93 g es per well ( p < 0.001 ) as compared with the control biofilm without es ( fig . 4a b ) . on ti , only 8.33 g es per well showed a reduction ( p = 0.005 ) of biofilm ( fig . biofilms of s. epidermidis formed on sss and pe also were reduced for all concentrations of es ( fig . biofilms decreased to minimums of 32.3% on sss for 25 g es per well ( p < 0.001 ) and 7.6% on pe for 8.33 g es per well ( p < 0.001 ) compared with the control . on ti , concentrations of 0.31 g es per well ( p < 0.001 ) and 0.93 g es per well ( p = 0.021 ) showed less biofilm ( fig . 4a cthese graphs compare the mean bf produced by s. aureus with es and the mean control bf after 8 days without es and answer the last research question whether es reduced biofilms produced by s. aureus . all values lower than the 8-day - old biofilm showed biofilm reduction . ( a ) the biofilm on sss was decreased for all concentrations with a maximum of 59.5% for 0.31 g es per well . ( b ) the biofilm on pe also was reduced for all concentrations with a maximum of 60.8% for 0.93 g es per well . ( c ) on ti , only 8.33 g es per well showed biofilm reduction . * for these comparisons all p values < 0.001 ; all other comparisons were similar.fig . graphs compare the mean bf produced by s. epidermidis with es and the mean control bf after 8 days without es and answer the last research question whether es reduced biofilms produced by s. epidermidis . ( a ) biofilm on sss was reduced for all concentrations with a maximum of 67.7% for 25 g es per well . ( b ) biofilm on pe also was decreased for all concentrations with a maximum of 92.4% for 8.33 g es per well . ( c ) on ti , only 0.31 g es per well and 0.93 g es per well showed biofilm reduction . * for these comparisons all p values < 0.001 ; all other comparisons were similar . these graphs compare the mean bf produced by s. aureus with es and the mean control bf after 8 days without es and answer the last research question whether es reduced biofilms produced by s. aureus . ( a ) the biofilm on sss was decreased for all concentrations with a maximum of 59.5% for 0.31 g es per well . ( b ) the biofilm on pe also was reduced for all concentrations with a maximum of 60.8% for 0.93 g es per well . ( c ) on ti , only 8.33 g es per well showed biofilm reduction . * for these comparisons all p values < 0.001 ; all other comparisons were similar . these graphs compare the mean bf produced by s. epidermidis with es and the mean control bf after 8 days without es and answer the last research question whether es reduced biofilms produced by s. epidermidis . ( a ) biofilm on sss was reduced for all concentrations with a maximum of 67.7% for 25 g es per well . ( b ) biofilm on pe also was decreased for all concentrations with a maximum of 92.4% for 8.33 g es per well . ( c ) on ti , only 0.31 g es per well and 0.93 g es per well showed biofilm reduction . * for these comparisons biofilm - associated infections related to implants can not be treated easily and frequently the implanted device must be removed with severe consequences for the patient . therefore , new approaches for these kinds of infections are needed . previously reported maggot es reduced bf of p. aeruginosa . in the current study , we investigated whether other bacterial species , namely , s. aureus , s. epidermidis , k. oxytoca , e. faecalis , and e. cloacae , were able to form biofilms on pe , ti , and sss , and if so , whether the quantity of bf differed between the bacterial species , whether the quantity of bf depended on the biomaterial surface , and whether the largest amount of bf was formed after 3 , 5 , 7 , or 9 days . finally , we hypothesized sterile maggot es could reduce bf on the biomaterials . first , we performed only a crystal violet assay as a standard method to measure biofilms , but other methods could be used in vitro and in vivo . we modified the crystal violet assay to our design with the combs of pe , ti , and sss , however the assay was based on the commonly used methods of pitts et al . and stepanovic et al . . second , every experiment was performed with one pool maggot es , but we did not compare the effectiveness between the various pools . the use of different pools will not likely influence the conclusions of this research , because in an earlier study , the effectiveness of es was compared and similar results of biofilm reduction occurred using various pools of es . for example , es were collected under standard conditions in darkness , had an acidity of ph 8 , and were incubated at 35c . furthermore , the protein concentration from each collected es was measured to standardize the various pools . the biomaterials were produced according to specifications for common implants for patients and all bacterial species were isolated from implant infections of patients in our clinic . therefore , we believe these results were representative and sterile maggot es reduce not only biofilms of p. aeruginosa but also existing and mature biofilms of s. aureus and s. epidermidis . these bacterial species were expected to form biofilms , as they all were isolated from device - related infections . the possible biofilm - forming capacities of the five tested species on medical devices have been described by others [ 4 , 10 , 20 ] . bf by k. oxytoca , e. faecalis , and e. cloacae was insufficient to be quantified . nutrient media other than bhi were used to test bf by these bacteria but did not show bf . the highest amount of biofilm was formed by s. aureus and s. epidermidis , which are the most frequently isolated pathogens from orthopaedic surgical wounds . . described equal bf but a stronger adherence to material surfaces by s. epidermidis . the quantity of bf depended on the biomaterial surface , however published studies show differing results . . found that the kind of metal , ti or sss , plays a minor role in the quantity of bf , whereas mackintosh et al . reported biomaterial surface characteristics we found ti had the least amount of biofilm in vitro formed by s. aureus and s. epidermidis . in a previous study , therefore , we conclude that the quantity of bf depends on the biomaterial and on the bacterial specie creating the biofilm . after 7 days of bf , the highest amount of biofilm was formed . in the first 7 days , the nutrient medium in the microtiter plate was not changed , because the biofilm bacteria were still viable as the biofilm kept growing . new nutrient medium with or without es was added after 7 days of incubation to maintain the viability of the bacteria . the average time for biofilm maturation is 6 to 8 days [ 10 , 22 ] . although the 7-day - old biofilms in this study were not examined by confocal microscopy , their maturation process was more advanced than that of the previously tested 24-hour - old pao1-biofilms which were decreased by es . thus , this research shows more evidence for the therapeutic potential of es for treatment against biofilm - associated infections which involve mature biofilms . the biofilm - decreasing capacity of maggot es seems optimal in low protein concentrations from 0.31 up to 8.33 g es per well ( 3.1 to 83.3 g es per ml ) . in previous studies , protein concentrations of as much as 20 g es per well were reported as the most effective concentrations for biofilm reduction [ 6 , 28 ] . there are several possible explanations for the fact that the influence of es at higher concentrations than 20 g es per well declined . we hypothesize a cofactor necessary to regulate the process was depleted or there is a nonenzymatic interaction . in future studies concentration ranges less than 0.31 g es per well will be tested to examine the lower limit amount of es causing biofilm reduction . previous research suggests maggot es do not possess direct bactericidal or bacteriostatic activity against planktonic organisms . therefore , es are not expected to reduce biofilm by destroying the bacteria in the matrix . however , the influence of es on biofilm bacteria will be investigated in future studies , by examination of total viable counts of bacteria . maggot es broke down existing biofilms , because all measurements were lower than the control bf after 7 days . however , they also seem to inhibit additional growth of bf as es reduced bf to less than 100% in the experiments with a greater amount of biofilm on day 8 compared with day 7 ( fig . 4a b ) . therefore , potential therapeutic use of es could be prevention or inhibition of bf , eg , flushing surgical wounds before closing and treatment against existing biofilm - associated infections , especially on orthopaedic medical devices . further research is necessary to clarify the mechanism(s ) of biofilm reduction by maggot es and identify the substance(s ) in es responsible for these reductions .

backgroundbiofilm - associated infections in trauma surgery are difficult to treat with conventional therapies . therefore , it is important to develop new treatment modalities . maggots in captured bags , which are permeable for larval excretions / secretions , aid in healing severe , infected wounds , suspect for biofilm formation . therefore we presumed maggot excretions / secretions would reduce biofilm formation.questions/purposeswe studied biofilm formation of staphylococcus aureus , staphylococcus epidermidis , klebsiella oxytoca , enterococcus faecalis , and enterobacter cloacae on polyethylene , titanium , and stainless steel . we compared the quantities of biofilm formation between the bacterial species on the various biomaterials and the quantity of biofilm formation after various incubation times . maggot excretions / secretions were added to existing biofilms to examine their effect.methodscomb-like models of the biomaterials , made to fit in a 96-well microtiter plate , were incubated with bacterial suspension . the formed biofilms were stained in crystal violet , which was eluted in ethanol . the optical density ( at 595 nm ) of the eluate was determined to quantify biofilm formation . maggot excretions / secretions were pipetted in different concentrations to ( nonstained ) 7-day - old biofilms , incubated 24 hours , and finally measured.resultsthe strongest biofilms were formed by s. aureus and s. epidermidis on polyethylene and the weakest on titanium . the highest quantity of biofilm formation was reached within 7 days for both bacteria . the presence of excretions / secretions reduced biofilm formation on all biomaterials . a maximum of 92% of biofilm reduction was measured.conclusionsour observations suggest maggot excretions / secretions decrease biofilm formation and could provide a new treatment for biofilm formation on infected biomaterials .

Introduction Materials and Methods Results Discussion

PMC3562671

age - related white matter lesions ( wml ) mainly affect information processing speed and executive function and entail an increased risk for cognitive decline and disability . in a meta - analytic study , high hazard ratios were also reported for incident stroke ( 3.3 ) and dementia ( 1.9 ) , but results from studies on wml association to dementia subtypes are inconclusive [ 46 ] . the prevalence of wml increases with age , and in a population study , 51% of randomly selected healthy subjects aged 4448 had wml . for the age range 6064 , all had wml , and 49% of these had at least one large ( > 12 mm ) wml region . in magnetic resonance ( mr ) imaging , white matter hypointensities in t1 weighted images , and white matter hyperintensities in t2 weighted and flair images are regarded as visualizations of wml . the conditions for demarcation of wml are enhanced in flair images due to suppression of the signal from free fluid . in contrast to t2 and t1 weighted images , this suppression causes fluid filled cavities in flair images to be hypointensive and excluded from wml by the intensity definition . however , the intensity suppression in the flair sequence entails incident imaging artifacts in the border region between wml and free fluid , for example , in the periventricular region . wml visible in mr imaging reflect demyelinization , axonal loss , gliosis , or edema . it is customary to use ct or mr imaging of wml among the diagnostic criteria for subcortical vascular dementia , for example , in erkinjuntti et al . . however , standardization of wml estimation is needed in order to establish uniform diagnostic criteria in clinical practice . visual rating is simple and fast and therefore an important candidate method for standardized clinical use . there are several visual rating methods for wml research purposes [ 11 , 12 ] . among these , the fazekas visual rating [ 13 , 14 ] is frequently used in research , and it has been shown to have good reliability compared to two other rating scales , but the results on its correlation to volumetrical assessment diverge . for example , the fazekas visual rating has shown the highest and lowest correlation to volumetrical assessment compared to other visual rating scales . manual volumetrical assessments have shown higher reliability than visual rating scales [ 11 , 15 ] and would be valuable in clinical settings if made less labour intensive . in gouw , visual rating of wml and wml volumetry had similar correlations to neuropsychological performance , but in garrett et al . , a correlation to neuropsychological performance was only found using wml volumetry and not in visual wml rating . several segmentation and thresholding techniques have been used to manually assess wml volume in the literature , but one of the few techniques with a methodological description is reported in gurol et al . . freesurfer is one of these methods , and it contains automatic assessment of neuroanatomical subregions as well as wml hypointensity volumes . freesurfer has been frequently used for neuroanatomical subregional volumetry and has been shown to be comparable in accuracy to manual labeling for many tasks [ 22 , 23 ] and to perform well compared to other automated segmentation tools . however , few publications have reported freesurfer wml volumes at all , and only one study reports intermethod reliability figures . one study found that total wml was significant for ad versus controls , and another found that total wml predicted functional decline almost as well as the best predicting regional wml regions . reliability analysis is the first step in assessing the accuracy of a wml method , and the often reported excellent or near excellent reliability may be a reason why methodological issues about reliability have not always been given enough attention . the aim of the present study was to compare three types of assessment methods of total wml in a clinical sample in order to examine different aspects of their reliability and to determine features in need of further methodological development . the manual mricron wml volumetry method was included on the basis of a presumed superiority in accuracy . freesurfer wml volumetry was included because of the automatic assessment of wml as well as the need for further validation because of the common use of the method . in the gothenburg mild cognitive impairment ( mci ) study , subjects between 40 and 86 years of age ( mean 65.6 , sd 7.7 years ) with subjective or objective cognitive impairment were recruited from the memory clinic at the sahlgrenska university hospital . exclusion criteria were acute somatic disease , severe psychiatric disorder , pseudodementia , and substance abuse or confusion caused by drugs . controls were recruited from other medical studies and senior citizen organizations , and baseline exclusion criteria were subjective or objective signs of cognitive disorder . the study was approved by the ethics committee of the university of gothenburg , and the subjects gave their informed consent to participate in the study . the study subjects were biannually assessed and classified according to the global deterioration scale ( gds ) . mci subjects remaining in the gds 2 to gds 3 range at followup were classified as mci stable , while mci subjects receiving a gds 4 ( mild dementia ) or higher classification at followup were classified as twenty eight controls , 69 mci stable , 9 mci converting , and 46 dementia subjects were included in the present study . wml was measured on the first 1.5 t mr imaging acquisitions in the study , between the years 2005 and 2007 . axial t2 weighted images were used for the fazekas rating , coronal flair images were manually segmented in the mricron software , and coronal t1 weighted images were analyzed with the freesurfer package ( stable release version 4.0.5 ) . in table 1 , the scan parameters used in the study are presented . all raters and operators were blinded for header data like identity and cognitive status of the subject . this study report was outlined to be in accordance with the guidelines for reliability studies in kottner et al . . periventricular wml and deep wml were not rated separately , in accordance with the recommendations by fazekas et al . , but only three grades were used as in inzitari et al . in contrast to the original four - graded fazekas scale , and in the present study , grade 1 also included possible absence of wml . for each subject , the slice with the largest wml occurrence visible was used to determine the wml load as belonging to one of three grades . the assessments were performed independently by two raters ( j. berge and j. eldblom ) who compared the images with the template images in axial orientation of the different grades of wml given by . the raters j. berge and j. eldblom had substantial knowledge of neuroanatomy and went through limited training in visual rating before entering the study assessment . cerebral wml segmentation and intensity thresholding were performed on 1.5 t coronal 5 mm flair images using the mricron software with a modification ( see later ) of the method used in gurol et al . . the segmentation method involves an initial rough manual demarcation of wml , separating them from noncerebral regions and septum pellucidum , as in holland et al . , within the same intensity span , followed by an intensity threshold set to separate wml from adjacent tissue types . in the gothenburg mci cohort , artifactual image intensity differences between slices and between series ( using the same flair sequence ) were common . no automatic intensity normalization between patients was done , but in order to consistently analyze each patient 's series in similar intensity settings , the mricron grayscale mapping was used on a window setting containing all brain tissue in a certain slice ( containing the quadrigeminal plate ) . due to intensity inhomogeneities , the manual volumetry method adapted from gurol et al . required a modification towards a more wml - specific manual segmentation , where the contour of the wml was demarcated quite closely . one rater ( e. olsson ) demarcated 152 subjects , while a second rater ( j. berge ) independently demarcated 27 randomly selected subjects for the determination of interrater reliability . the rater e. olsson has longstanding experience in mri segmentation , and the rater j. berge went through substantial training in the manual wml volumetrical method before entering the study assessment . hypointensity volumetrics determined as wml were estimated by an operator ( n. klasson ) running the freesurfer analysis ( stable release version 4.0.5 ) . freesurfer is a highly automatic image analysis suite and is available for download online ( http://surfer.nmr.mgh.harvard.edu/ ) . freesurfer uses a probabilistic atlas generated from manually segmented mr scans to execute the segmentation . the probabilistic information has been mapped into talairach space so that each location therein contains specific probabilities for each tissue type . given a specific location and tissue type , the probabilities are given as ( 1 ) a gaussian intensity distribution , ( 2 ) probability of occurrence , and ( 3 ) probability of neighboring tissue types . during segmentation , a number of processes take place including motion correction and intensity normalization , removal of nonbrain tissue , talairach registration , and labeling of voxels into tissue types . an initial labeling takes place where each voxel is assigned its most probable tissue type . an iterative algorithm then uses the found tissue probabilities to calculate new probabilities for the voxel labels . manual edits were done by the operator ( n. klasson ) to reduce inaccuracies in white and grey matter classification . the distribution of wml volumes in the gothenburg mci study was right skewed , and normality could not be assumed , not even after log transformation . the reliability analyses comprised spearman correlation , intraclass correlation ( icc ) ( with two - way mixed , absolute agreement and single measurements model ) , and kendall 's tau . differences in reliability between the highest wml tertile and the lower tertiles were tested with fischer 's r to z transformation . for differences between raters and methods , the study characteristics for the controls , mci stable , and dementia groups are presented in table 2 . dementia patients had significantly lower mmse scores than all other groups , had significantly lower education than stable mci , and were significantly older than stable mci . all three wml assessment methods detected significant associations between wml estimate and age ( fazekas 0.385 , manual mricron 0.334 , automatic freesurfer 0.524 ) . regarding interrater reliability , the spearman rho coefficient was 0.89 with regard to the modified fazekas visual rating scale and 0.60 regarding the manual mricron wml volumetry . the rho value was 0.65 for the intermethod correlation between freesurfer automatic wml volumetry and manual wml volumetry ( table 3 ) . no significant systematic differences between raters were found for fazekas visual rating or for mricron manual volumetry in the wilcoxon matched pairs test ( details not shown ) , but the freesurfer automatic volumetry differed significantly from the manual mricron volumetry . almost all freesurfer volumes were lower than the corresponding manual mricron volume ( figure 1 ) . in order to evaluate reliabilities in the dense and the sparse parts of the data distribution ( figure 2 ) , respectively for the aggregated lower two tertiles , the fazekas interrater reliability ( spearman 's rho ) measured 0.65 , and for the upper wml tertile 0.92 , this difference was significant . the manual mricron interrater reliability was nonsignificant for the lower two tertiles ( which make up about 10 percent of the whole volume range ) but significant for the upper wml tertile with an interrater reliability of 0.94 . for the anterior part of the brain taken separately , there was still no significant reliability for the lower aggregated wml tertiles , but it was significant for the posterior part with a rho value of 0.56 . for the intermethod reliability between the manual mricron volumetry and the automatic freesurfer volumetry , the rho value was also lower , 0.38 in the lower aggregated tertiles , than in the upper tertiles , 0.74 . for icc values , the reason why we do not report them in the text is stated in section 4 . figure 3(a ) shows the bland - altman ( ba ) plots of absolute volume interrater differences in the manual mricron method , demonstrating increasing differences with higher volumes . however , the variation in rater differences was larger in the assessments of the lowest range of the wml volumes embracing about 80% of the subjects . when comparing the difference as a percentage of the mean measures of each subject ( figure 3(b ) ) , the variation was even more pronounced with regard to the lowest part of the wml volumes , while there was no clear indication of an increase in rater differences with larger volumes . the intermethod ba plots ( figure 1 ) for the manual mricron volumetry and automatic freesurfer volumetry showed a similar pattern as the interrater ba plots , with a pronounced variation for low wml volumes in figure 1(b ) . it is common to evaluate reliability in wml research with intraclass correlation ( icc ) with excellent results , for example , in gao et al . and smith et al . however , it is misleading to use such an analysis in a data structure where the distribution is skewed , with very sparse data points in the upper third of the volume range . while showing an excellent reliability for the whole sample , like previous studies , the icc in the lower aggregated tertiles for the manual method was nonsignificant ( table 3 ) . the density of low - burden wml ( figure 2 ) probably represents a very common clinical distribution , and reliability analysis must under these conditions be performed and interpreted with caution . the finding in the present study of lower reliability in the fazekas rating with lower wml burden is congruent with the finding by wardlaw et al . where cohorts with lower wml burden showed lower reliability . the higher reliability for high wml burden has been considered as a ceiling effect , but there might as well be floor effects in visual rating . presumed ceiling and floor effects do not disqualify visual rating as a candidate for clinical use , but such effects would in general lower the usefulness for wml research , for example , the possibility to find valid correlations with psychometrics . the nonsignificant spearman correlation in the aggregated lower tertiles between manual mricron volumetry raters could possibly be due to the high density of data affecting the rank order of the ratings . it is unclear if the high spearman correlation for high - burden wml implies an overestimation of the reliability due to the sparse data density ( leading to less error with ranked data ) or if there really was a higher reliability as the ba plot of fractional differences seems to imply ( figure 3(b ) ) . in our opinion , the low reliability in low - burden wml is most probably not due to the combination of high density of data and the nature of the spearman correlation . rather , it may be a real rater- and intermethod variation as is visible in the ba plots which seem to indicate an increased variation in the lowest quarter of the volume range . the higher rater variation in low wml may in turn be due to difficulties in the handling of intensity distortion affecting the thresholding step in the manual volumetry . intensity inhomogeneities in mr images and variation in grayscale level between scan series result in inconsistent classifications of hyperintensities that in turn introduce measurement errors in the manual wml assessment . in order to assess wml volumes as accurately as possible under conditions with varying intensity levels through the image slices of a subject , a methodology was chosen where the thresholding was adjusted as a compromise to best fit the visible wml volume through all image slices of a subject 's brain . since the localization and extent of wml vary , the accuracy of the thresholding can be expected to be decreased by these intensity distortions . a particular shift in grayscale level was observed in the anterior part of all flair image series ( figure 4 ) , which may be due to gradient eddy currents or cross talk between 2d flair slices [ 34 , 35 ] . the unreliability for low wml seems to emanate mainly from the anterior half of the image data , and the intensity shift in the anterior part may well be a reason for more complex considerations in the thresholding of low wml burden cases . for higher wml volumes , the thresholding step in general only affects the amount of wml selected , but for lower volumes , the thresholding more often affected the presence or absence of wml in a slice , which means a more straightforward thresholding with higher volumes . in short , increased complexity in the thresholding may be the main cause of an increased rater variation for low wml volumes . the freesurfer suite includes intensity normalization steps which to some extent will limit intensity distortion , but that also may extinguish some of the hypointensities . further , the t1 mr sequence used for the freesurfer volumetry does in general have lower wml definition and somewhat smaller areas of wml hypointensities compared to the hyperintensities in the flair sequence . a visual inspection of the t1 weighted images and the freesurfer segmentations confirmed that the substantial deviations between the volumetrical methods in the detection of wml in various locations may mainly be due to the lower definition in the t1 weighted images . the freesurfer segmentation often omitted large amounts of deep wml seen in the flair images ( figure 5 ) . on few occasions , occasionally , freesurfer detected more wml in the periventricular region than visible in the t1 weighted images . compared to wml seen in flair images , the amount of wml found by freesurfer still was lower even in the periventricular regions . the t1 mri images in figures 5 and 6 are examples showing that the noise level makes the detection of punctate wml a difficult task , nevertheless freesurfer detects several punctate wml patches in this region . the single punctate wml patch in the flair slice in figure 6 is hardly visible in the t1 slice , and it is conceivable that no punctuate wml patch in this location is detected in freesurfer . in some cases , freesurfer detected small punctate wml not detected in the manual volumetry ( figure 6(b ) ) , but occasionally , it detected less punctate wml than the manual volumetry ( figure 6(c ) ) . however , a more accurate punctate wml detection may be a disadvantage in dementia research since punctate wml often originate from perivascular spaces and have been found to have low progression . in short , the freesurfer volumetry generally detects less wml , which is likely due to a combination of the method , the characteristics of wml , and the visibility of wml in t1 weighted images and leads to larger differences between the methods when measuring larger wml volumes ( figure 1 ) . wml progression generally begins in the periventricular region and subsequently expands radially to more peripheral locations . the skewness of the distribution of wml , with a low fraction of high - burden wml subjects also in the dementia part of the sample , can be expected to exist also for clinical samples in general . the statistical atlas used in freesurfer was generated from a small sample ( http://surfer.nmr.mgh.harvard.edu/fswiki/buckner40testing ) , where high - burden wml cases might be rare or lacking . an atlas generated with a low frequency of high - burden exposure may contribute to the freesurfer inability to find deep wml . the fazekas rating was performed by comparing the largest wml aggregation to the templates , and no metrical measurements were used which possibly affected the classification of the subjects close to the fazekas definitions of the metrical borders . the visual rating in three grades differs from the design in other studies , which limits the comparability of results . it is not feasible to use a zero grade wml in t2 images with any accuracy , and in a later control of the flair images , it was ascertained that no subjects with complete absence of wml were present in the study . in visual rating , the flair sequence can be expected to be advantageous compared to the t2 sequence due to the signal suppression of free fluid in flair images . however , the flair sequence in this study had coronal orientation and 5 mm slice thickness , which made it unsuitable to use with the axial template images . the basal ganglia nuclei in flair images often had no clear intensity separation from white matter , which may have contributed to an inaccurate volume assessment in this region . the hyperintensities along the ventricular lining in the basal ganglia region were included in the manually assessed wml , which may be unsuitable and possibly confound the differentiation , primarily in patients with low wml volumes . the different scan sequences used in the different methods confound the methodological comparison ; for example , only the flair sequence does to some extent show separate intensity ranges for fluid filled cavities and wml . by contrast , in t1 and t2 sequences , fluid - filled cavities have intensities in the same range as wml . the moderate interrater correlation for the manual volumetry could possibly be a consequence of intensity distortions in the flair images . although steps were taken in the thresholding methodology to minimize the impact of intensity distortions , sample tests of wml volume using different thresholdings unveiled considerable variation in measurements . the flair sequence had obvious intensity inhomogeneities , and no automatic intensity normalization was performed which may limit the possibilities to interpret the present findings further . reliability analysis showed acceptable overall results but with lower reliability for all methods in the lower aggregated tertiles . despite excellent overall reliability for manual volumetry hence , the results of intraclass correlation in wml samples , that is , commonly skewed , might be misleading , and reliability analysis of wml methods should be considered with caution . optimized mr imaging , postscan intensity standardization and normalization are quite likely among the most important factors to consider for more accurate measurements of wml . freesurfer comprised lower volumes than the manual method , probably due to the t1 sequence it uses , and was not able to detect punctuate wml in a consistent manner . a medically oriented follow - up paper focusing on the predictive power of the different wml measurements is being prepared . in another study , we intend to refine the visual comparison between the methods , using a larger sample of subjects and coregistration of images from different scan series . finally , our long - term goal is an improved manual method that excludes thresholding but includes an adequate intensity normalization .

age - related white matter lesions ( wml ) are a risk factor for stroke , cognitive decline , and dementia . different requirements are imposed on methods for the assessment of wml in clinical settings and for research purposes , but reliability analysis is of major importance . in this study , wml assessment with three different methods was evaluated . in the gothenburg mild cognitive impairment study , mri scans from 152 participants were used to assess wml with the fazekas visual rating scale on t2 images , a manual volumetric method on flair images , and freesurfer volumetry on t1 images . reliability was acceptable for all three methods . for low wml volumes ( 2/3 of the patients ) , reliability was overall lower and nonsignificant for the manual volumetric method . unreliability in the assessment of patients with low wml with manual volumetry may mainly be due to intensity variation in the flair sequence used ; hence , intensity standardization and normalization methods must be used for more accurate assessments . the freesurfer segmentations resulted in smaller wml volumes than the volumes acquired with the manual method and showed deviations from visible hypointensities in the t1 images , which quite likely reduces validity .

1. Introduction 2. Method 3. Results 4. Discussion 5. Conclusions

PMC2755304

salts of monovalent ions are highly soluble in aqueous solution , and the solvated ions have important functional roles in screening charge , moving charge , and also influencing the structure and dynamics of biomolecules such as proteins and nucleic acids . given this , accurate modeling of biomolecular structure , dynamics , and function requires some representative model of ionic interactions and mobile ions . in this work , we focus on the most common molecular mechanical models , specifically those based on a pairwise additive potential that includes a coulombic treatment of the electrostatic interactions and a lennard - jones representation of dispersionattraction and core repulsion . in this formulation , the potential energy ( uij ) between any pair of nonbonded atoms ( i and j ) in a system composed of the ions and water molecules is usually expressed as here , qi and qj are the point charges of the atoms , rij is the distance between atoms , and rmin , ij and ij are the van der waals radius and well depth of the lennard - jones potential . to represent the water , simplified point charge models such as tip3p,(8 ) spc / e,(9 ) and tip4pew(10 ) are utilized . although the models are simple , determination of the lennard - jones parameters for the ions is fairly subtle , and therefore , a wide variety of different parameter sets have been proposed . many of these are summarized in our previous work that introduced a new set of force field parameters for the alkali ( li , na , k , rb , cs ) and halide ions ( f , cl , br , i ) where the specific choice of lennard - jones parameters depends on the specific choice of water model.(11 ) here we further investigate the structural and dynamic properties of these ions in comparison with other existing parameter sets or force fields . new properties calculated include activity coefficients , diffusion coefficients , populations of ion clusters , residence times of ionion and ionwater dimers , association constants , and solubility . although many of these properties have been determined experimentally , some properties ( such as ionwater residence times and ion clusters populations ) are elusive or disputed and can only be estimated through simulation . we compare our results with existing experiment and previous simulations , noting that much of the prior simulation work is fragmental as it tends to focus on calculating the properties themselves rather than assessing the influence of the force field . the intent here is to provide insight into the validity and range of applicability of simple pairwise atomic molecular mechanical models of alkali and halide ions . this information can help guide the development of more accurate models of ions in aqueous solution . assuming there are only solute molecules a and solvent molecules b in a molecular system , the chemical potential a , is as follows where xa , a , k , and t refer to the mole fraction , activity coefficient of molecule a , boltzmann constant , and temperature , respectively.(20 ) the chemical potential in the standard state composed of pure a molecules is a0 . equation 2 can be rewritten in more convenient molality units ( ma [ mol / kgsolvent ] ) instead of mole fraction(21 ) where m0 = 1 mol / kg and the new activity coefficient in terms of molality ( a , m ) is related to the original activity coefficient through the relation , a , m = axb . contributions to the activity term ( ktln a , m ) can be estimated by subtracting chemical potentials calculated at different concentrations . this is not trivial since , as the concentration rises ( up to 1 molality ( m ) ) , the experimentally measured activity coefficients for nacl and kcl drop down to 0.6 , which is equivalent to a very small energetic change in the chemical potential ( 0.30 kcal / mol at 300 k ) . at low concentrations , due to the sensitivity of the chemical potential to changes in the activity coefficient , only very small errors in the calculated chemical potential are tolerable . as typically errors in calculating free energies ( or the chemical potential ) in conventional simulations are around 1 kcal / mol in most practical applications,(22 ) reducing the error to the order of 0.01 kcal / mol essentially requires an enormous number of ensembles or , equivalently , much longer simulations . to overcome this problem , previous investigations estimated activities using the potentials of mean force acting on the counterions . the benefit of this approach is that the explicit water is no longer necessary once the potentials of mean force are constructed . to more fully simulate the relevant ensemble , we chose instead to use fully explicit simulations following kokubo and co - workers.(21 ) chemical potentials can be calculated by applying the widom method(23 ) as expanded into ntp ensembles by shing and chung.(24 ) when a test particle of a is inserted in the system with na 1 particles of a(solute ) and nb particles of b(solvent ) , the chemical potential of a can be expressed as where a and qa are thermal de broglie wavelength and the molecular partition function of particle a ; v is the volume of the system ; = 1/kt ; and is the potential energy of the test particle interacting with the rest of the pre - existing atoms . equation 4 includes two terms as shown below , and the excess term can be simplified assuming the correlation between energy and volume is weak . the excess term is equivalent to the solvation free energy of the test particle , which can be determined by several methods . in this research , we chose to use thermodynamic integration ( ti ) . in order to calculate activity coefficients , equate eq 3 and eq 4 and rearrange to obtain activity coefficients go to 1 as the molality goes to zero thus , the reference state of the most diluted solution ( ) can be introduced subtraction of eq 7 from eq 8 gives the calculation of each term in eq 9 is straightforward , except for the last term , which is the difference of the solvation free energy of the inserted particle at a specified concentration from that of the most diluted solution . as mentioned , previous attempts to calculate mean activity coefficients of alkalihalide salts were limited to implicit solvent methods using the potential mean force between molecules . lyubartsev and laaksonen derived the potential mean force using smith and dang s nacl(27 ) in flexible spc water,(28 ) while gavryushov and linse used spc / e water and aqvist(29 ) and dang s parameters . lenart(14 ) used koneshan and rasaiah parameters(30 ) for van der waals interactions of ions , and the potentials in water were adjusted for a specifically designed implicit solvent model . experimental methods to measure mean activity coefficients are various , but the results are all very similar.(31 ) we used the values summarized the crc handbook(32 ) for comparison . the more intuitive method is direct calculation of the average mean square displacement of ions for a unit time period . calculation of the velocity autocorrelation function also provides a link to the diffusion coefficient.(15 ) however , given long simulations , the two methods give equivalent results for the diffusion coefficients . in this research information regarding the dynamics of ions in water is very limited and relies mostly on simulation . for example , the correlation time ( or the residence time ) of ionwater and ionion pairs can easily be calculated by simulation . previous estimates of the residence times applied the autocorrelation function designed by impey et al.(15 ) more recently , laage and hynes raised the issue that residence times calculated by that method are very sensitive to the tolerance time.(33 ) the tolerance time was originally introduced to the calculation in order to represent the transition state between the paired ( reactant ) and unpaired ( product ) states as the reactant state moves to become the product state . in other words , this catches the case when a pair only briefly becomes unpaired before pairing again , with a time period less than the tolerance time . the updated method by laage and hynes suggests calculating the new time correlation function ( r(t ) ) based on the stable states picture model.(34 ) to apply this method , the states of stable reactant , r , ( or paired ) and stable product , p , ( or unpaired ) need to be defined . in the r state , molecule pairs are separated by almost the radius of the first shell ( rmax1 ) , which is the first peak of their radial distribution function ( rdf , g ) . the two molecules in the pair reaches the p state when they are separated by almost the radius of the second shell ( rmax2 ) , which is the second peak of the rdf . the probability , p(t ) , is the probability that a molecule pair in r state at time 0 transforms into the p state after time t : the residence time ( ) of the molecule pair is defined by the equation above . although experimental measurements of the solubility of alkalihalide crystals in water are easily found , it is not simple to calculate solubility in simulation . to compare with experiment , to do this , the chemical potential of the crystal and solutions at various concentrations is calculated and the concentration at which the two potentials are equal indicates saturation concentration . instead of this more tedious approach , we used a more direct method to derive the equilibrium state . sufficiently large crystals in contact with an almost saturated ion solution will reach an equilibrium state given sufficient sampling . to avoid finite size surface effects of a small crystal , we note that this approach , although direct , will likely require longer simulations to fully equilibrate the heterogeneous mixture . however , the direct method is advantageous since there are no underlying assumptions ( as are required in the calculation of chemical potentials ) . as a result molecular dynamics simulations were performed with the sander module of amber9 using the amber nonpolarizable potential energy function shown in eq 1 . general conditions applied in all of the molecular dynamics simulations , unless otherwise stated , are discussed below . a 2 buffer was built into the pairlist , and rebuilding of the list was triggered if any particle moved more than 1.0 . long - ranged electrostatic interactions were estimated with the particle mesh ewald method , and a homogeneous density approximation was applied to correct for the cutoff of the lennard - jones interactions . particle grids for the ewald summation were approximately one grid point per angstrom in each dimension . the atomic charges for the grid points were smoothed onto the grid using a fourth - order b - spline . temperature and pressure were regulated by using weak coupling algorithm.(41 ) the coupling time for temperature and pressure control was 1 ps . the target temperature and pressure were 298 k and 1 bar , respectively . all of the water models used here are rigid , and the shake algorithm(42 ) was applied to constrain all bonds to hydrogen with a tolerance of 10 . the initial coordinates of water and ions were randomly positioned in a rectangular periodic box , which was prepared to have density of 1 g / cm . the initial box size was about 3840 , and the number of waters and ions in each case is displayed in table 1 . the systems were minimized by steepest descent for 1000 steps , followed by a two - step equilibration . in the first step , md simulation was performed for 40 ps in the nvt ensemble . in the second step , a cationanion pair was inserted in the system slowly to calculate the hydration free energy of the ion pair . lennard - jones interactions of the inserted ion pair were turned on . in the second step , the charge of the anion was activated . the perturbation steps adopted the following mixing rule of the potential energy : u( ) = f()u0 + ( 1 f())u1 , where u0 and u1 are the potential energy of the initial state and the final state . intermediate states between perturbations were determined adjusting the parameter , ( 0 1 ) . for the first perturbation , f( ) = ( 1 )i = 06(6 + i)!/6!/i ! , and for the second and third perturbations , f( ) = 1 was applied , respectively . numerous intermediate states between perturbations were defined by substituting with these numbers : 0.01 , 0.05 , 0.1 , 0.15 , 0.2 , 0.25 , 0.3 , 0.35 , 0.4 , 0.45 , 0.5 , 0.55 , 0.6 , 0.65 , 0.7 , 0.75 , 0.8 , 0.85 , 0.9 , and 0.95 for the first perturbation and 0 , 0.05 , 0.1 , 0.15 , 0.2 , 0.25 , 0.3 , 0.35 , 0.4 , 0.45 , 0.5 , 0.55 , 0.6 , 0.65 , 0.7 , 0.75 , 0.8 , 0.85 , 0.9 , 0.95 , and 1 for the second and the third perturbations . using the previously equilibrated coordinates , all of the states were simulated in the ntp ensemble to calculate < du / d>. the initial 100 ps for each of the simulations was discarded . the < du / d > values were fit into a cubic spline as a function of , and this was used to calculate 01du / dd numerically , which is equivalent to eq 6 . shown are the molalities ( m ) of kcl and nacl solutions simulated when calculating the activity coefficients , including the number of explicit ion pairs ( nion ) and water molecules ( nw ) in the periodic unit cell . the initial coordinates of the water and ions were randomly placed in a rectangular periodic box keeping the density of the system to be 1 g / cm . fifteen hundred explicit water molecules and 27 ion pairs were added to make the concentration 1 m. the box sizes were approximately 3640 . note that lif was omitted since its solubility is lower than 1 m.(32 ) each system was minimized and equilibrated in the same way as described in the previous section , and then md simulations were performed for 10 ns . each curve was smoothed using a bzier curve to better obtain the characteristic radii of the rdf curves at dg / dr = 0 . the three shortest radii were determined , and they were assigned sequentially as the radius of the first shell ( rmax1 ; radius of the first peak ) , the radius of first coordination shell ( rmin1 ; radius of the first minimum after rmax1 ) , and the radius of the second shell ( rmax2 ; radius of the second peak ) . the first shell was refined by repeatedly fitting the curve into quadratic equations.(11 ) the radius at the vertex of the quadratic fit was substituted for rmax1 . the relationship , 6d = limtd < r>/dt was utilized to calculate the diffusion coefficient ( d).(44 ) average mean - square displacement ( < r > ) of each type of molecule up to 10 ps was plotted versus time every 1 ps . the curves were fit to a straight line that passes through the origin by least - squares . the diffusion coefficients were then corrected for finite periodic box size effects as per yeh and hummer.(45 ) this was done by measuring the diffusion coefficients of ions with various sizes of the system , specifically 1500 water molecules and 27 ion pairs , 1000 water molecules and 18 ion pairs , and 500 water molecules and 9 ion pairs . these simulations were extended to 10 ns each . for the analysis of ion clusters , cluster populations were analyzed from the simulation of 1500 water molecules and 27 ion pairs . calculation of the residence time for a paired molecule requires the precise determination of the r and p states , which are based on the distance of the two molecules . the upper boundary of the r state ( rr ) was the radius satisfying g(rr ) = (g(rmax1)g(rmin1 ) ) . a pair of molecules at the same or shorter distance than rr was considered to be in the r state . in a similar way , the lower boundary of the p state ( rp ) was determined by the radius satisfying g(rp ) = (g(rmin1)g(rmax2 ) ) . a pair of molecules separated by a distance equal or larger than rp were defined to be in the p state . originally , the lower boundary of the p state suggested by laage and hynes was the radius corresponding to the half - energy barrier in mean force potential curve . however , our definition of the boundary is equivalent to their definition because radial distribution is exponentially proportional to the potential of mean force ( i.e. , g(r ) e , where w(r ) is the potential of mean force ) . the residence time , , was calculated by integrating the time correlation function eq 10 . because r(t ) can not be evaluated infinitely , the equation was integrated only from 0 to 1 ns and the rest of the integration was carried out under the assumption of exponential decay : integration of r(t ) over t was performed using the trapezoidal rule , and eq 12 was numerically solved to obtain . rectangular crystals of alkalihalide salts were built using their lattice constants as described previously.(11 ) when building the nacl - type crystals , the unit crystal was aligned exactly parallel to the rectangular axes ( figure 1a ) . on either side of the yz planes of the crystals , an ion and water mixture with the density of 1.52.0 g / cm was prepared . presaturated ion solutions were composed of roughly 20% more ion pairs than expected to be soluble in terms of molal concentration ; 862 ( licl ) , 266 ( nacl ) , 206 ( kcl ) , 336 ( rbcl ) , 901 ( libr ) , 397 ( nabr ) , 246 ( kbr ) , 304 ( rbbr ) , 534 ( lii ) , 531 ( nai ) , 386 ( ki ) , 336 ( rbi ) , 1626 ( csf ) ion pairs and 2000 water molecules . for the cscl - type crystals ( cscl , csbr , and csi ) , the unit crystal was initially aligned to the rectangular axes then rotated 45 along the z axis in order to reduce the net dipole along the x axis ( figure 1b ) . this prevented the strong net dipole from artificially breaking the crystal immediately after the start of the simulation . on either one of the yz planes of the crystals , an ion and water mixture with the density of 1.52.0 g / cm was apposed ; 120% saturated solutions and various other solutions at different concentrations were tried . however , we encountered several problems with cscl - type crystals as explained in the next section . coexisting salt crystal and ion solution in a periodic box . the horizontal axis is the x axis , and ion solution was placed on one of the yz planes of the crystals . ( a ) nacl - type crystal and ( b ) cscl - type crystal . the equilibration and subsequent dynamics were normally not very sensitive to the precise setup of the crystal solution and its initial density . however , in a few cases , the crystal instantaneously disintegrated and melted into the aqueous solution due to instability in the built crystal ; in these cases , the results were discarded . as the simulation cell is periodic , the initial setup constituted infinite crystal sheets with aqueous ion solution between the crystal sheets . the system was minimized by using the steepest descent method for 1000 steps and equilibrated at a constant temperature and pressure for 40 ps while harmonically restraining the coordinates of crystal ions to their initial positions with a force constant of 50 kcal / mol / . pressure was controlled by adjusting the periodic box size anisotropically so as not to break the connectivity of the crystals between neighboring boxes . that is , pressure applied to x , y , and z directions were calculated separately , and the three edges of the box were rescaled according to the three normal pressures . anisotropic rescaling intrinsically requires longer equilibration time , but it is indispensible in nonisotropic systems like this . the coupling time for the pressure regulation was 1 ps . after removing the restraints , md simulation was continuously performed and the crystals gradually grew or dissolved as they moved toward the equilibrium state . the distribution of the water and ions along the x axis changes as the crystal grows or is dissolved . to obtain the distribution of molecules at different times in a consistent manner , a static reference needed to be defined . on the time scale of the md simulations performed , the inner crystal layers remained intact . therefore , we used the geometric center of the inner crystal layers as the reference point for the x axis distribution . from the reference point , the histogram of the molecules along the x axis was collected up to half of the x axis edge of the periodic box ( xmax ) with a step of 0.1 . this final histogram was converted to number density per unit volume by dividing by the volume of each bin of the histogram . the bulk molar concentration of the molecules in the solution was assumed to be the average molar concentration of the molecules located between xmax 10 and xmax from the reference point . the volume and total potential energy of the system were traced and averaged . the system was considered to reach the equilibrium state when both the volume and potential energy leveled off . to avoid introducing biases when determining the equilibrium state , running averages over 60 ns of the two properties were recorded every 100 ps along with their standard deviation . the ranges that these averages cover with the tolerance of their standard deviations were compared each other . when the average overlap of the ranges from the last 40 ns onto the very last range is above 95% , we considered the system to be in equilibrium state ( see figure s1 in the supporting information ) . the final concentration of the solution phase was considered to be the average concentration of the last 30 ns . activity coefficients of nacl and kcl solutions at varying concentrations ( 5 m ) were calculated with tip3p water(8 ) and the tip3p - compatible ions . only this limited set was investigated as these simulations were very costly and effectively required the equivalent computer time of 11 700 days 4 processors based on the performance of available clusters . yet , these results still provide general insight into how much the activity coefficients from simulations vary with respect to experiment . to calculate activity coefficients , the number of explicit atoms in the periodic box was adjusted to simulate a variety of concentrations , as is shown in table 1 . in eq 9 , all of the other parameters were determined ( see table 1 ) except for the excess chemical potential . as the activities are very sensitive to small changes in the excess chemical potential terms , it is necessary to discuss how the error was estimated . in general , the errors of < du / d>i from the ith ti window can be estimated if the correlation length ( c ) of < du / d>i is known.(46 ) given this , we denoted the error as ew , i , and it was estimated using the relationship where ( rmsd)i is the rms deviation of < du / d>i and ti is the length of the simulation required to obtain the rmsd . although we did not precisely estimate the correlation length ( c ) of < du / d>i for all of the ti windows , we expect that the correlation lengths are shorter than 1 ps ( for example , please refer to the decay of the autocorrelation curve shown in figure s2 in the supporting information ) . when we assume that the correlation lengths are less than 1 ps , the error estimates should be valid . as the simulations proceeded , it was noted that some windows had inherently low errors , dropping below 0.00001 kcal / mol within a few hundreds of picoseconds of simulation , whereas other windows required tens of nanoseconds before the error drops below 0.1 kcal / mol . the contribution of each window to the free energy change of a perturbation is roughly inversely proportional to the total number of windows ( nw ) of the perturbation because the ti windows are almost evenly distributed . therefore , an error in free energy calculation of each perturbation ( ep , j ) is where the subindex j denotes jth perturbation . finally , the error of the total free energy calculation ( etotal ) can be estimated as we differentiated the time of simulations for the higher concentrations ( > 1 m ) and the lower concentrations ( < 1 m ) because we wanted higher precision at the lower concentrations . the finally estimated total errors fell around 0.082 kcal / mol for the higher concentrations and 0.034 kcal / mol for the lower concentrations . however , real errors might be even smaller because we assumed that c was 1 ps , which is likely an overestimate . the calculated excess chemical potentials are listed in table s1 and displayed in figure 2 . in order to calculate activity coefficients of ion pairs applying eq 9 , the difference in chemical potential from the most diluted solution due to the practical difficulty in carrying out simulations of high volume systems , with explicit solvent , we did not simulate solutions at less than 0.037 m. in fact , this is not sufficiently dilute , and we could not assume that the activity coefficient of the solution is unity at this concentration . when they are compared to experimental activity coefficients,(32 ) the chemical potential at this concentration is almost 0.1 kcal / mol more negative than that from the most diluted solution ( i.e. , 0.1 kcal / mol ktln m ) . , we assumed that there is 0.1 kcal / mol difference in chemical potential at 0.037 m from the most diluted solution . in actuality , the values of the activity coefficients at the other concentrations are greatly affected by this number . however , choosing this number is reasonable , and regardless of the precise value , the overall shape of the curve of activity coefficients at various concentrations is preserved . the activity coefficients calculated with this assumption are listed in tables 2 and s1 . calculated ( calc ) and experimental(32 ) ( ref ) mean activity coefficients of nacl and kcl versus square root of molality . the x axis denotes the square root of molality of an ion pair , and the y axis indicates its mean activity coefficients . closed circles and closed squares denote the activity coefficients of tip3p - compatible ion pairs , nacl and kcl , in tip3p water . open circle and open squares are the experimental mean activity coefficients of nacl and kcl in water . the contributions to the chemical potential from the three perturbations are indicated as vdw , elec1 , and elec2 ( table s1 in the supporting information ) . the vdw generally increased as the concentration of the solution increased for both nacl and kcl . for elec1 , the insertion of sodium ions was less favored as concentration increased ; however , it became more favored for potassium ions beyond 1 m. overall , the contributions to the differences in the chemical potentials observed were relatively small ( columns 2 , 3 , and 4 in table s1 in the supporting information ) with the exception of the hydration free energies . the calculated activity coefficients deviate from the experimental values as the concentration increases ( table 2 ) . however , the curve of nacl was placed above that of kcl , which qualitatively corresponds to the experimental curves . although we will discuss solubility further below , the solubility of nacl and kcl determined by simulations in tip3p water was actually lower than their experimental solubility . however , we did not observe crystal formation on the time scale of these simulations , suggesting the solutions were supersaturated above certain concentrations . lyubartsev and laaksonen calculated the activity coefficients of smith and dang s nacl(27 ) in flexible spc water.(28 ) the results are very impressive as their numbers are very close to the experimental mean activity coefficients , even up to 5 m. both observations imply that potential energies among smith and dang s na , cl , and spc / e water are reasonably balanced and can properly estimate the activity coefficient and the solubility . however , this does not sufficiently support the idea that all of the smithdanggarrett ions are appropriately balanced . we note that it is also possible that the flexible spc water model provides better effective interaction potentials than spc / e . gavryushov and linse applied a similar methodology to lyubartsev and laaksonen , except that they used spherical boundary conditions and the spc / e fixed - point water model . they determined mean activity coefficients of aqvist s ions(29 ) ( na and k ) in addition to smith and dang s ions . note that the activity coefficients of nacl generally correspond to the true activity , regardless the source of the ion parameters , while those of kcl tend to poorly reproduce the experimental activity coefficients . this likely results from inaccurate estimates of the solubility of kcl in simulation with available parameters compared to experiment . lenart et al.(14 ) simulated ion solutions with a specially designed implicit solvent model that included a distance - dependent dielectric permittivity , and this led to improved results . however , it should be noted that they did not use a standard combining rule for the lennard - jones potential and instead defined separate lennard - jones interactions between all of the atom pair types . this suggests that current pair potentials still have room for improvement in the context of doing away with the simple combining rules . experimental and computed diffusion coefficients of alkali and halide ions at infinite dilution are listed in table 3 and compared with those from pre - existing ion models . the diffusion coefficient increases as the size of ion increases with characteristic peaks at rb and br . although previous work claims to reproduce the qualitative trend of the diffusion coefficients with the smithdanggarrett ion parameters , we were not able to reproduce those results . we only found the characteristic peaks with the water - model - specific cation parameters in spc / e . the differences likely relate to differences in how the long - range electrostatic interactions were determined and the correction for the finite size effects . moreover , in the current work , larger periodic unit cells were applied , which may also partially explain the difference . until our recent work , yet , it is well - known that tip3p water diffuses too rapidly and this in turn leads to ions that are too swift in tip3p water . consequently , experimental diffusion coefficients are better reproduced in tip4pew and spc / e water , although there still are significant discrepancies . these discrepancies partially result from concentration effects as the simulations were performed in 1 m solution while the experimental numbers are from dilute solution . when the concentration of the ions in solution increases , the diffusion coefficients of the ions decrease.(18 ) diffusion coefficients of the ions also appear to depend on the choice of counterion as ions tend to move swifter when they are solvated with fast - moving counterions ( see table s2 in supporting information ) . generally , if the counterion is larger , the increment in the diffusion coefficient is greater . considering the relative diffusion rates of the ions and water molecules , swift molecules tend to pull or push the ions and thus enhance the diffusion coefficients of the ions . also , spc / e - compatible br diffuses most rapidly with k and rb . looking at the residence time between cation and anion ( shown in the next section ) , the results suggest that high residence times impede the movements of both the cations and anions ( because the paired ions are effectively heavier ) . generally , the larger ions have higher diffusion coefficients ; however , the relative values are influenced by ion pairing and the diffusion rates of other molecules in the solution . diffusion coefficients were calculated at 1 m concentration with the counter ions cl and na . note that the experimental diffusion coefficients(32 ) represent dilute solution . the diffusion coefficients before correcting for finite size effects are also provided in the supporting information . the population of these clusters ( displayed in table s4 in the supporting information ) is closely related to the other dynamic properties of the solution.(51 ) from the data , the association constant for each single cationanion pair was calculated . the true association constant is the ratio of the activity of ion pair to the activities of each of the single ions ( k = apair/(acation aanion ) ) . due to the overwhelming computational demands of estimating activities for each of the ion models , we assumed the activities were unity and substituted mole fraction , molar concentration , and molal concentration . the association constants in terms of molar concentration are similar and are reported in the supporting information . chen and pappu(52 ) performed a similar analysis using qvist ions(29 ) where the association constants were calculated in terms of molal concentration . when these numbers are compared with fuoss conductimetry results,(53 ) the results suggest that the combination of qvist cations with the anions from chandresekhar et al.(54 ) and lybrand et al.(55 ) is excellent . however , this analysis neglected the activity coefficients . as shown in chen and pappu s table 3 , the association constant is clearly greater when concentration is low , which proves activity coefficients can not be ignored . the association at the low concentration is indeed close to the actual association constant because activity coefficients are close to unity at low concentrations . if ion - dipole effects are neglected , the activity coefficients of ion pairs are almost unity . if we take experimental mean activity coefficients for single ions from nacl and kcl , they are 0.779 and 0.768 at 0.1 m. therefore , 1.65 ( = 1/0.779 ) for nacl and 1.70 ( = 1/0.768 ) for kcl should be multiplied to their calculations . in other words , association constants of water - model - specific ions , and smithdanggarrett ions are shown in table 4 , and generally the values were far less than fuoss association constants even though these numbers are probably underestimated . fuoss estimated the numbers based on the conductometric data assuming that the paired ions play no effective role in conductance of the ionic solution . however , consistent with chen and pappu , we assume that the distances between ion pairs are within rmin1 . however , gurney radii from fuoss data ( see fuoss table 2 ) , which are arguably the criterion determining free ion and ion pair , are generally much greater than our rmin1 values , especially for li- , na- , and k - associated salts , although the distances for some rb- and cs - associated salts are slightly shorter than our rmin1 values . for this reason , his nonconductive ion pairs possibly include some solvent - shared pairs as well as contact ion pairs . the association constants were recalculated using the criteria provided by fuoss to see how much the association constants change depending on the criteria ( table s5 in the supporting information ) . although significant differences from fuoss estimation were still observed , the new association constants agreed much better with fuoss numbers . with the same assumptions , most of the association constants of qvist s ions calculated by chen and pappu would exceed fuoss estimation . it is not clear why the qvist ions , compared to the other ion models , show such significant differences . ion concentrations listed in table s4 of supporting information were used to calculate the constants in terms of both molal concentration . the unit of the constants is m. the deviations were calculated from the deviations of the individual populations in table s4 of supporting information . the residence time between two molecules is an indicator of the lifetime of pair association at a particular temperature . we calculated residence times of cationanion , watercation , and wateranion pairs in 1 m solutions using water - model - specific and smithdanggarrett ion models . the characteristic radii from the rdf were used in the determination of the residence time and are listed in the supporting information ( table s3 ) . the residence times between li and f are considered to be very long ( compared to the md simulation time scale ) , and thus we could not estimate their values . also , we could not calculate the residence time between tip4pew - compatible cs and f and smithdanggarrett cs and f in spc / e water because of an insufficient size for the ensemble . consistent trends in the cationanion residence times across a particular column or row of the table ( as a function of the ion sizes ) are not observed . when the cation is fixed ( along the column in table s6a in the supporting information ) , the curves of the residence times generally monotonically increase , monotonically decrease , or display a convex shape with a single maximum . for the tip3p - compatible cations li and na , the residence times monotonically drop as the size of the paired anions increases . k and rb have maximal residence time with br , whereas cs does with i. the trend is similar with the tip4pew - compatible cations . however , here na has a maximal residency time with br , while k , rb , and cs have their peaks with i. spc / e - compatible cations behave slightly differently from the ions of the other models . na ( and possibly li ) ions have both local minimum and maximum with cl and br , respectively . however , smithdanggarrett cations do not have such maxima in the middle of the series . when the cation is smaller than na , cationanion residence times continuously decrease as the size of the anion increases . when the cation is larger than k , residence times tend to increase as a function of the anion size . these behaviors consistently can be observed in the three different types of water models . on the other hand , in the series of anions ( along the row in table s6a in the supporting information ) , the curves of the residence time generally show two patterns : monotonous decrease or concave shape having one minimum . the minimums are located at slightly different positions depending on the anion and ion models . all the tip3p - compatible anions have the longest residence time when they are paired with li . the residence times in the series gradually decreased as the size of the pairing cations increases excluding i , which has a slight increase compared with cs . however , in the series of br and i , the shortest residence times were reached at rb . minimums of residence times also can be observed with all the spc / e - compatible anions ; f with k , and the other anions with na . for the smithdanggarrett anions , such minimums unexpectedly , the residence time of i with these parameters in spc / e shows an increasing pattern as a function of cation size . overall , the residence times are complicated and depend on both the ion parameters and the applied water model . residence times between cations and water molecules ( table s6b in the supporting information ) show little dependence on the size of the counterions . unlike cationanion residence times , the average watercation residence times generally are not strongly sensitive to the choice of ion force field , although this appears to be less true with li and na . however , the differences among anionwater residence times with different ion models are small , even with the smallest anion , f ( table s6c in the supporting information ) . these observations are confusing because , while significant influence of the water and ion parameter model is observed for anioncation interactions , little is observed with ionwater residence times . this likely results from the heavy focus on ionwater interactions , as compared to ionion , in force field development . although our work in the development of water - model - specific ion models did include ionion interactions , specifically , the crystal lattice energy and lattice constant , the ionion residence times lack consistency . on the other hand , residence times of ionwater pairs ( wi ) are closely related with the diffusion coefficients of ions ( di ) and water molecules ( dw ) . this can be seen when di and dw/wi1/2 are plotted in an ad hoc manner to show relationships among the data ; when this is done , a roughly linear correlation is observed ( figure s3 in the supporting information ) . interestingly , the slopes of the linear relationship are almost identical no matter what ion or water models are used . however , the diffusion rates clearly depend on the concentration of the ions and also the nature of the cationanion and waterwater interactions . unexpected crystallization of alkalihalide salts at concentrations below their saturation limit motivated our development of new ion parameters.(11 ) a necessary test of these parameters is alkalihalide pair solubility . transitions in the crystal structures , detected by sudden change of volume or potential energy , were observed . also , the structures of the newly growing crystals on the surface of the pre - existing crystal were different from their natural crystal structure . another problem was exposed in salts paired with fluoride ions . the volume and the potential energy of the systems fluctuated at low frequency . although the systems may satisfy the condition of equilibrium as described in the methods section , in this case , the equilibrium states were suspicious because of the low - frequency fluctuations of the two properties . for these reasons , we only show the valid solubility results in table 6 . depending on the choice of ion pair and ion parameters , simulations required about 110530 ns for the solution crystal coexisting systems to reach their equilibrium states . this simulation time could , in principle , be reduced if the initial concentration and density of the solution opposed with the crystal are very close to its saturation condition . notwithstanding , it is presumed that at least 100 ns of simulation are required . the equilibrium concentrations of the cation and the anion in the same system were almost identical as expected . thus , the concentrations of the paired salt shown in table 6 are the average of the cation and the anion . the chemical potentials of crystals can be assumed to be constants because the dependency of chemical potential of crystal on its size is usually negligible . the low solubility of the tip3p - compatible ions implies that the chemical potential of the crystals are too low in general because at low concentration the deviation of the activity coefficients from the experimental results is small , as shown in figure 2 , and the deviation of chemical potential of the solution from the real solution might be small . although the absolute solubility values of the tip3p - compatible ions deviate significantly from experiment , the qualitative solubility trends are most accurately reproduced with these parameters compared to the other models ( table 6 ) . smithdanggarrett ions also showed low solubility in tip3p water , except for nai , which had a higher solubility than true nai solution . however , the qualitative trends were not observed with smithdanggarrett ions in tip3p water : nacl and nai were relatively good , but the other salts showed significant deviation . tip4pew - compatible ions well reproduced the saturation concentrations with na - associated or cl - associated salts . spc / e - compatible ions showed similar behaviors with na - associated or cl - associated salts , but the solubilities were not as accurate as tip4pew ions . smithdanggarrett ions did not behave much differently in spc / e water than they did in tip3p water . they showed relatively good solubilities with nacl and nai . since the ion models are identical for the smithdanggarrett ions , the chemical potential of the crystals are presumably the same in both water models . in addition to this , as the solubility is not strongly affected by the water models , the chemical potential of the solution as a function of concentration is likely also almost identical regardless of the choice of water model . as a result , we can presume that the activity coefficients do not change greatly as a function of concentration in different water models as long as the ion models are identical . however , absolute hydration free energies can be different.(11 ) the various simulation sets except tip3p their solubilities were generally well reproduced with nacl . however , as the relative distance from nacl becomes farther , the deviation of the solubilities seems to increase . to better understand the deviations in solubility compared to experiment lattice energy is the potential energy difference generated when ions in a crystal are separated to infinite distance . a plot of the difference in experimental and calculated lattice energies versus the natural log of the ratio of calculated and experimental molal concentrations exposes trends for the different ion model parameter sets ( figure 3 ) . if good agreement between the experimental and calculated values in both properties is obtained , the points will tend to cluster about the origin . the data suggest that the errors in the lattice energies for the various salts calculated using smithdanggarrett ions are generally positive ( calculated < experiment ) in contrast to water - model - specific ions.(11 ) interestingly , regardless of the sign of the deviation in the lattice energy , most of the data points are located above x axis . the data in the figure also suggest that greater absolute deviations between experiment and calculation of the lattice energy tend to lead to greater deviations in the solubility . however , there are trends in the deviations of the lattice energy that depend on the choice of cation . with the water - specific ion parameters , na - associated salts were less sensitive to the deviation of lattice energy ( solid line ) while rb - associated salts were much more sensitive ( dashed line ) . although there is a slight discrepancy regarding the extent of the effect , the solubility of smithdanggarrett salts was similarly affected by the deviations of lattice energy , and the effects were also distinctive depending on the cations . in conclusion we tried to find correlations of solubility with lattice constants , but we could not find obvious correlations . these results suggest that the parametrization of the tip3p - compatible ions can be revisited to improve salt solubilities by focusing more heavily on reproducing the lattice energy . x axis indicates the difference of the lattice energy calculated ( lecalc ) from the experimental lattice energy ( leref).(11)y axis indicates the logarithmic ratio of experimental saturation concentration ( mref ) to the calculated saturation concentration ( mcalc ) in terms of molality . each point denotes the alkalihalide salts , as shown in the legend on the right - hand side . different colors specify the ion and water models : blue for tip3p - compatible ions in tip3p water , magenta for tip4pew - compatible ions / tip4pew water , yellow for spc / e - compatible ions in spc / e water , cyan for smithdanggarrett ions in tip3p water , and violet for smithdanggarrett ions in spc / e water . among them , water - model - specific ions were fit to y = ax curves , where a is a fitting parameter . the solid line , the dotted line , and the dashed line are the fit curves for na - associated salts , k - associated salts , and rb - associated salts . figure 4 shows the correlations between the association constants of alkalihalide pairs ( listed in table 4 ) and their solubility . generally , the two properties were inversely proportional , such that ion pairs with higher association constants have a greater tendency to form crystals . fuoss association constants do not show this correlation ( figure 4b ) ; however , as discussed , their gurney radii might not be able to distinguish contact ion pairs from solvent - shared pairs , which means the reported association constants also include non - crystal - forming associations . in any case , the correlation between the calculated cluster populations and the saturation concentrations is useful since the more easily calculated association constant can be utilized to estimate solubility . correlation of the association constant ( k ) of cationanion pair to the saturation concentration ( m ) . calculated molal saturation concentrations of salts are inversely proportional to the association constant of the ion pair ( a ) . the solid line indicates the fitted curve , mcalc = 0.593k ( r = 0.7618 ) . however , the experimental saturation concentrations(32 ) did not show correlation with fuoss association constants(53 ) ( b ) . the results suggest that high cationanion residence times drive the ions in solution toward the crystal form , whereas high watercation and wateranion residence times tend to dissolve the crystal ( see the legend of figure 5 ) . although the results appear reasonable , these observations can not be experimentally validated as we do not have an experimental measure of the residence times . correlation of the residence times to the saturation concentration . under the assumption that the inverse of residence time is proportional to the dissociation rates , and that the pairing between ionwater and ionion is a first - order reaction implying that the association constants are the ratio of the rates of the forward and backward reactions , the calculated molal saturation concentrations of alkalihalide salts ( mcalc ) were plotted against c - a/w - a w - c . here c - a , w - c , and w - a are residence times of cationanion , watercation , and wateranion , respectively . the solid line indicates the fitted curve of all the data points , y = 0.460x ( r = 0.7656 ) . the color scheme is same as that in figure 3 . by investigating these correlations , the links among the properties including diffusion coefficients , residence times , association constants , and solubility figure s3 in the supporting information shows that the diffusion coefficients of the ions are largely dependent on the choice of water model . also , looking at the smithdanggarrett ions in table s6b , c in the supporting information , the ionwater residence times also depend on the choice of water model . the dependency of the residence times on the choice of water model was generally in the order , wateranion < watercation < cationanion , as shown in table s6 in the supporting information . when we assume that the wateranion and watercation residence times can be correctly calculated , this suggests a critical need to balance the ionion interactions . this balance between the residence times is also closely related to the solubility , as shown in figure 5 , and even to the association constants based in figure 4 . originally , water - model - specific ion models were optimized using multiple properties reflecting ionion interactions . in spite of the effort , cationanion residence times , association constants , and solubility show large variability depending on the choice of water model . this , along with the sensitivity of the solubility to the lattice energy ( figure 3 ) , again emphasizes the importance of ionion interactions in the parametrization of ion force fields . the relevance of each of these properties ( diffusion coefficients , residence times , lattice energy , association constants , and solubility ) to the activity coefficients was not assessed in detail here as the limited set of calculations performed was extremely computationally demanding . the activity coefficients of the smithdanggarrett ions calculated by gavryushov and linse(12 ) suggest that the calculated nacl activity coefficients are relatively good compared to kcl . these results suggest that , if solubility is accurately simulated , the activity coefficient should also be accurately reproduced . therefore , the tip3p - compatible ions should not be used for simulations at high salt concentrations . although smithdanggarrett ions are fine with nacl , their kcl is not appropriate for simulating high salt environments . for this reason , we recommend tip4pew and spc / e water models together with their water - model - specific ions for simulating high salts concentration . the dynamic and energetic properties of alkali and halide ions were calculated in simulation using various different ion and water force fields including our previously developed water - model - specific ion parametrizations.(11 ) mean activity coefficients were determined for nacl and kcl in tip3p water . qualitatively , the mean activity coefficients correspond to experiment ; however , the deviations tend to increase as the salt concentration increases . although extremely computationally demanding , to better determine which model is most appropriate for reproducing the experimental activity coefficients , all of the pairs of alkalihalide salts should be investigated using explicit solvent with the various parameter sets . however , these and previous results do clearly suggest that accurate estimation of solubility is critical to accurately calculate activity . diffusion coefficients appear to be largely dependent on the choice of water model . generally , faster moving water models resulted in higher diffusion coefficients for the solvated ions , but at the same time , the diffusion constants were almost inversely proportional to the square root of the residence time between the ion and the water molecule . we also calculated the association constants of various alkalihalide salts in terms of molar and molal concentration . however , the lower association constants could be explained by two reasons , specifically ( 1 ) since fuoss experimental data use a greater cutoff length defining ion pairs which may include solvent - separated pairs , and ( 2 ) if activity coefficients are properly considered , our calculated association constants in terms of molar and molal concentration would increase . the residence times between ion and water and ion and ion were also calculated . ionion residence times varied significantly according to the choice of water model , while the ionwater residence times did not . the tip3p - compatible ions showed qualitatively correct differences among the various salts , but the solubilities were generally significantly underestimated . the tip4pew and spc / e ion parameter models well reproduced the solubilities of na - associated salts and cl - associated salts . smithdanggarrett ions also qualitatively reproduced the solubilities of nacl ; however , the deviations of the other ions in the parameter sets from experimental were generally greater than observed with the tip4pew- and spc / e - compatible ion parameters . some of the dynamic and energetic properties of alkali and halide ions show a strong dependence on the choice of water model . ion force fields can then be developed on top of that foundation with care to balance the ionion interactions as many pairwise properties such as the association constant , the cationanion residence time , and the solubility are very sensitive to these . in summary , the results suggest that our spc / e and tip4pew water - model - specific ion parameters fairly accurately model the behavior of the monovalent ions in aqueous solution across a wide range of concentrations , whereas the tip3p - compatible ion parameters should be avoided at high salt concentrations .

the dynamic and energetic properties of the alkali and halide ions were calculated using molecular dynamics ( md ) and free energy simulations with various different water and ion force fields including our recently developed water - model - specific ion parameters . the properties calculated were activity coefficients , diffusion coefficients , residence times of atomic pairs , association constants , and solubility . through calculation of these properties , we can assess the validity and range of applicability of the simple pair potential models and better understand their limitations . due to extreme computational demands , the activity coefficients were only calculated for a subset of the models . the results qualitatively agree with experiment . calculated diffusion coefficients and residence times between cationanion , watercation , and wateranion showed differences depending on the choice of water and ion force field used . the calculated solubilities of the alkalihalide salts were generally lower than the true solubility of the salts . however , for both the tip4pew and spc / e water - model - specific ion parameters , solubility was reasonably well - reproduced . finally , the correlations among the various properties led to the following conclusions : ( 1 ) the reliability of the ion force fields is significantly affected by the specific choice of water model . ( 2 ) ionion interactions are very important to accurately simulate the properties , especially solubility . ( 3 ) the spc / e and tip4pew water - model - specific ion force fields are preferred for simulation in high salt environments compared to the other ion force fields .

Introduction Methods Results and Discussion Conclusion

PMC4969700

a primary goal of treatment development for serious mental illness ( smi ) is to reduce the disability commonly associated with this type of psychiatric diagnosis . disability associated with smi imposes significant personal and economic burdens across multiple domains of life activity . schizophrenia and schizoaffective disorders are among the leading global causes of disability . including care expenditures and disability , the costs of smis are more than $ 317 billion annually in the united states or more than $ 1000 per year for every man , woman , and child . many areas of functioning are affected by smi , including activities of daily living ( adls ) , vocation , finances , social and family relationships , leisure activity , and health care . even when successful for improving some aspects of illness , such as positive symptoms , treatment with medication can fail to improve or restore functioning . cognitive impairment and negative symptoms there have been multiple recent efforts to develop novel compounds designed to improve cognition and negative symptoms in schizophrenia and the functional disability associated with these illness attributes . with respect to cognition , the us food and drug administration has indicated that improvements in the results of neuropsychological testing would be insufficient for a medication to receive an indication for improving cognitive function in schizophrenia . it has long served as the standard in alzheimer disease research , with varying levels of success . similarly to schizophrenia , assessment of functioning in alzheimer disease is complicated by factors such as heterogeneity of the illness and stage of the disease . assessments appropriate for measuring function in moderate or severe stages of the illness are not able to detect the subtle expressions of the illness in the very early stages . this has become an increasingly important issue in alzheimer disease research , given a more recent focus on developing early intervention strategies . coprimary measures incorporating caregiver report to assess functioning in mid- and late - stage alzheimer disease have been more successful , given the high degree of contact caregivers have with these patients . even with the limitations of informant reporting , availability of informants for the alzheimer disease population differs greatly from that of schizophrenia , where informants are often unavailable and tend to have less frequent contact with patients . although coprimary measures have not been required for studies of investigational compounds designed to address negative symptoms , these studies have incorporated measures of functional outcome as important secondary outcome measures . assessing functional outcome in a reliable and valid manner for studies of cognitive impairment and negative symptoms we discuss issues that are particularly relevant for such studies and describe several recently developed novel approaches to assessment , which may address some of these issues . table i provides a list and brief description of various assessment measures referenced in the following sections . a particularly daunting issue with respect to the assessment of functioning in smi is the breadth of potentially relevant outcome domains . functional outcomes can refer to independent living skills , including paying bills , basic self - care , taking care of one 's living space , social skills , social network size , work and academic role functioning , and the ability to complete goals . to further complicate the enterprise , functioning in each of these areas selection of measures can be difficult , particularly in illnesses such as schizophrenia in which clinical presentation and functional impairments can be heterogeneous . these issues are further complicated by a proliferation of available measures purported to assess functional outcomes . as one example of the proliferation of measures of functional outcome , the valero study ( validation of everyday real - world outcomes ) asked 48 experts to nominate measures , resulting in 59 potential functional assessments for further examination . in another study that examined social functioning measures , a review of 301 studies identified 80 potentially relevant measures of social functioning . given the proliferation of measures , it is not hard to understand why some may question the need for novel measures . in addition to the difficulties in selecting from the vast array of measures , functioning means different things to different stakeholders . practitioners , individuals with lived experience of smi , families , and payers may prioritize various types of functional outcome very differently . careful thought must guide the development and use of these pros to ensure that these outcomes are relevant to patients and other stakeholders . it is also unclear what should be expected of measures of functional outcome in terms of time course for change . in contrast , some changes in real - world functioning may take many months or years . green has recommended a focus on more proximal or intermediate measures of functioning in short - term clinical trials rather than a focus on real - world functional changes , such as increased number of friends , active participation in dailylife , or work or vocational performance . examples of intermediate measures include those that assess functional capacity , such as the university of california san diego ( ucsd ) performance - based skills assessment ( upsa ) or brief international functional capacity assessment ( bifca ) , formerly known as the functional assessment battery ( fab ) , and those that are interview - based measures of functional impairment associated with impaired cognition , such as the cognitive assessment interview ( cai ) . intermediate measures such as those mentioned above are designed to be more sensitive to change in the context of a brief clinical trial , but a number of limitations associated with these measures remain unresolved . interview measures typically rely on self - report or collateral information from a caregiver informant . functional capacity assessments that require the performance of specific functional tasks in front of an examiner ( eg , calling a doctor 's office to schedule an appointment ) are informative regarding how well a person can perform a functional behavior in a research setting , but they offer little data regarding what the person is doing in their day - to - day environment . any information about an individual 's day - to - day functioning can only be inferred from these types of intermediate measures . a large number of functional outcome measures , including intermediate measures such as the cai , rely primarily on the report of the person being assessed . the person is asked to report on how well they get along with others ; how often they engage with others socially ; how well they perform independent living skills ; the quality of their work ; and to what extent problems with attention , memory , or planning interfere with these activities , etc . a number of problems with self - report data have been identified . difficulties with memory over long periods of time ( usually the past week , month , or 3 months ) can render retrospective self - report of activities inaccurate . this is particularly problematic in an illness such as schizophrenia where individuals score nearly two standard deviations below control subjects on measures of memory . this may impact how the person views their competence at specific tasks , whether the person is aware of cognitive impairment and its consequences in daily life , who they consider to be friends , and how they view the quality of their work . a recent study by harvey et al ( valero ) found that patient ratings on the specific levels of functioning scale ( slof ) were not well correlated with cognitive impairment and other ratings of functional outcome . on the other hand , harvey et al found that ratings by caregivers and ratings by a trained clinician using all sources of information were correlated with both cognitive test scores and with other functional outcome assessments . these data indicate that for some individuals , self - report is unlikely to produce reliable data . using caregivers to provide information on functioning greatly it is not clear how much time a person may need to spend with a participant or how much access to the person 's residence would be needed to be able to report reliably as a caregiver . in the valero study , informants were typically case managers who saw patients at least once per week . the inability to identify an appropriate informant to supply rating information has been reported by as many as one - half to one - third of patients diagnosed with schizophrenia . in clinical trials , requiring the participation of caregivers can often increase the length of time to recruit participants and the cost of the study . in addition , requiring caregiver participation may have substantial consequences for the generalizability of findings , as it would result in the de facto exclusion of an entire participant subgroup unable to identify caregivers . functional capacity measures resolve the issue of who is reporting by changing the assessment to one based on objective task performance . in deciding who should report on functional outcome , the type of assessment needs to be taken into account . for example , with respect to reporting on adherence to medication , the correlation between patient - reported adherence over a 2-week period and objective measures such as in - home pill counts or electronic monitoring was close to zero . however , in a separate study , when patients were asked daily using a smart pill container to report their adherence , self - reported adherence rates were highly correlated with objective measures ( r=0.61 ; p<0.0001 ; study using med - emonitor devices for electronic monitoring ) . it seems that individuals with smi can be good reporters of what they do over short periods , but may do less well at reporting over long periods of time ( weeks ) and at identifying their level of competence in certain domains . we are particularly interested in discussing these developments as they pertain to studies in which cognitive impairment or negative symptoms are primary outcomes . in the next two sections we will describe assessment development in the area of functional capacity measures . then , we will go on to discuss assessment development for intermediate and real - world measures designed specifically for trials in which negative symptoms are a primary target . when assessing functional outcome , it is important to consider the construct that is being investigated and how this construct may affect functioning . for example , functional capacity measures are intermediate measures that are highly correlated with neuropsychological test scores ( r0.60 ) . consequently , it makes sense to use measures of functional capacity for studies in which change in cognition is a primary outcome . however , if negative symptoms are a primary study target , intermediate measures that assess motivation or effort in a laboratory setting or actual engagement in daily functional activities may be more appropriate to include . the upsa and its brief version the upsa - b have served as well accepted and validated measures to assess functional ability in individuals with smi . the upsa assesses functional performance across five subscales including household chores , communication , finance , transportation , and recreational activity planning . the full upsa takes approximately 30 minutes to administer , and upsa - b , which includes only the finance and communications subscales of the upsa , takes 10 to 15 minutes to administer . an important potential advantage reported for the upsa - b is its sensitivity to change in response to a treatment designed to improve functioning . the upsa is strongly correlated with cognitive functioning , and this relationship has been replicated across multiple countries . the test of adaptive behavior in schizophrenia ( tabs ) is a functional capacity measure designed to address limitations of other available measures , including inadequate assessment of the ability to initiate and of the ability to self - identify problems that occur in the course of performing functional activities . for example , capacity tests tend to ask a participant how they would solve a particular problem . however , it is not clear that the individual would have been able to identify the fact that a problem existed . in addition , since capacity tests tend to ask the individual to respond to a contrived situation , there is little room to assess the individual 's ability to initiate a response . the tabs is designed to assess the abilities necessary to perform goal - directed activity , including initiation , planning , problem identification , problem solving , sequencing , appropriate inhibition , and persistence in the context of six functional areas ( work and productivity , medication management , independent living , shopping , basic hygiene , and social skills ) . items in the tabs demand considerable initiation ( eg , spontaneously naming items that would be necessary to stock an empty bathroom ) , allow the subject the chance to identify specific problems on his or her own before the problems are pointed out by the examiner ( eg , identify that he or she was short - changed , identifying that he or she will run out of medication ) , and provide additional points for spontaneously offering solutions ( eg , spontaneously announcing a plan to remedy a problem with running out of medication ) . tabs scores were found to be moderately to strongly correlated with other measures of functional outcome , negative symptoms , and neuropsychological test scores ( convergent validity ) . a brief form of the tabs containing three subtests ( assessing medication management and work / productivity ) takes approximately 15 minutes to administer and score . as part of the matrics initiative ( measurement and treatment research to improve cognition in schizophrenia ) , the vim ( validation of intermediate measures ) study assessed the reliability , validity , and utility of a number of intermediate measures assessing functional outcome in schizophrenia . findings indicated that the upsa , upsa - b , and tabs were the instruments with the most favorable psychometric properties in a us sample . different cultures may influence the degree to which the activities investigated are relevant to individuals and subgroups in different countries . we therefore examined ratings made by experienced researchers at 31 sites in eight countries in order to assess the cross - cultural adaptability of three functional capacity measures including the independent living scales ( ils ) , upsa , and tabs . english - speaking research staff familiar with conducting medication trials rated the extent to which each subscale of each intermediate measure could be applied to their culture and to subgroups within their culture on the basis of gender , geographic region , ethnicity , and socioeconomic status . problems were identified for specific subscales on all the performance - based assessments across multiple countries . india , china , and mexico presented the greatest challenges in adaptation . in a follow - up study within the overall matrics initiative , the psychometric properties of the bifca , comprised of two subtests from the upsa and one from the tabs , were examined . these subtests were rated by clinical trials experts in the study described above to be the most appropriate functional capacity assessments across different cultural contexts in both western and non - western cultures . researchers administered the bifca , the upsa - b , the matrics consensus cognitive battery ( mccb ) and used other scales to assess measures of symptomatology and a measure of global functional outcome in 141 individuals with schizophrenia at baseline and four weeks later . the psychometric properties of the bifca and upsa - b were similar in india . given the previous results that the subtests on the bifca are more relevant to non - western cultures , the bifca has been translated into multiple languages for potential use in international studies of novel medications seeking an indication for improving cognition in schizophrenia . it is unclear whether this battery will be adopted as a coprimary outcome in future studies examining cognitive impairment in schizophrenia . identifying limitations in the representativeness of role - play scenarios for complex , real - life situations , the canadian objective assessment of life skills ( coals ) measure was developed to assess functional competence . the first involves the sufficient representation and engagement of component processes that make up a functional behavior and the sensitivity to capture it . the second involves the need for new measures to demonstrate incremental validity above neurocognitive tasks previously- shown to predict functional outcome . in the construction of the coals , mcdermid vaz et al incorporated multiple component processes ( eg , working memory , comprehension , and social cognition ) required to perform functional tasks into the development of the assessment tasks . an important feature of the coals development was the collaboration through focus groups including clinicians and peer specialists to ensure that all domains were covered and that constructs were well assessed . the coals provides a competence measure in two functional domains , including procedural knowledge routines ( eg , how to cook a meal ) and executive operations ( ie , knowledge and flexibility necessary to carry out tasks and successfully overcome challenges that may present during task performance ) . the coals consists of scenarios tapping into five domains , including health and hygiene , time management , transportation , crisis management , and domestic activities . the coals total scores and scale scores demonstrated good concurrent validity with measures of the upsa and mccb and good discriminant validity . the coals was also shown to add incremental validity by accounting for additional variance in the prediction of community independence over neurocognitive measures alone . as a recently developed measure , it has yet to be determined whether the coals provides a measure of functional capacity that is superior to existing measures . it is also unclear to what extent the coals could be adapted for use across cultures . in studies investigating novel compounds to improve negative symptoms , choice of functional outcome is an important issue . functional capacity , which may have little to do with increasing motivation , effort , and emotional responsiveness , may not be the ideal choice . at least two approaches to the development of intermediate or proximal measures for studies of negative symptoms have been applied . one examines laboratory measures of the expenditure of effort as a proxy for motivation , and another attempts to rate the amount or real - world activity engaged in by the research participant in multiple functional domains . it is possible that these types of intermediate measures would be ideally suited for studies in which negative symptoms are the primary clinical target . green and colleagues have done some elegant preliminary work on measures of effort - based decision making . basically , effort - based decision making examines how much effort one is willing to expend for a given level of reward . for example , someone is offered a choice between something that requires little effort and results in little reward and something that requires significantly more effort but offers greater reward . by manipulating the difficulty of the tasks and levels of reward , these tasks allow us to infer a person 's level of motivation . particularly , amotivation is thought to be associated with the willingness to expend less effort in such paradigms and has been associated with poorer functional outcomes . patients with schizophrenia have in fact been found to choose easier tasks over more difficult tasks in many of these paradigms . reddy and colleagues examined multiple measures of effort in the domains of perceptual effort , physical effort , and cognitive effort in 94 individuals with stable schizophrenia . these tasks were found to have low - to - moderate correlations with negative symptoms , cognitive functioning , and functional outcome measures , and were generally- not related to positive symptoms . their study provides preliminary support for further investigation of these effort - based measures as intermediate measures in trials investigating functional outcomes associated with negative symptoms . another way to assess more proximal functional outcome in studies of compounds designed to address negative symptoms is by assessing changes in activity in functional domains . the daily activity report ( dar ) examines real - world functioning in a unique way by obtaining a report from the patient of all activities over a 7-day period . the dar was developed following a comprehensive review of the literature , as well as from input in focus groups from patients , nonprofessional caregivers , and treatment team members . patients rated different activities for importance and complexity , forming the basis for the dar scoring system . the dar differs from an existing measure developed by jolley , which examines functional behavior over 7 days in a number of important ways . the dar is based on frequent contact with the patient ( three telephone calls daily for 7 days ) rather than a long period ( 1 week ) of retrospective recall . moreover , the dar uses a structured interview to rate activities and their complexity in three different domains ( domestic , social , educational/ occupational ) , with scoring based directly on the input of stakeholders . the data present a complete picture of what the person is doing during a week . the data can be summarized in multiple ways and can provide information that would allow a researcher to make ratings on global instruments assessing functional outcome , such as the personal and social performance scale ( psp ) . global scales are typically rated during an interview with the patient , and scores are either based on self - report of activities recalled over months or on the report of significant others . we administered the dar and additional assessments of functional outcome , functional capacity , cognition , and symptomatology to 50 individuals with schizophrenia at two time points , 1 month apart , and to 25 healthy controls . early indications are that the dar is well tolerated by patients , with 88% of those approached consenting to participate , and 94% of those who consented completing both the baseline and follow - up assessment phases . in addition , patients and controls did not differ in the number of calls completed during the 1-week assessment , with a mean number of 17.77 ( standard deviation [ sd]=3.83 ) and 18.32 ( sd=2.53 ) calls respectively . test retest reliability across 1 month for the total dar score was 0.67 ( p<0.0001 ) . the total dar score , as well as scores for social activity and nondomestic work / school , differed significantly between control and patient participants . dar domain scores were moderately - to - highly associated with negative symptoms and with functional outcomes . interestingly , the work / school dimension of the dar explained most of the variance in the dar 's relationship to global functioning . this relationship suggested that global measures of functional outcome might primarily be tapping whether the person works . work status is unlikely to change in a brief clinical trial and this may be why some of these global measures of functional outcome can be relatively insensitive to change . dar scores were only weakly and nonsignificantly related to positive symptoms , providing important evidence of discriminant validity . this study provided preliminary support for the reliability and validity of the dar using interviewer administration . current work is focused on testing a patient - reported outcome version of the dar , using sms texting technology . this version of the dar automatically scores activity on the basis of patient responses and eliminates the need for interviewers . such a system , which can capture all of a patient 's waking activity without the use of a research assistant , will probably be both highly informative and cost effective . these two approaches to functional assessment for studies investigating compounds to improve negative symptoms represent important moves forward however , it is important to point out that neither of these measures examines the emotional responsiveness dimension of negative symptoms . one approach to creating a performance - based measure of emotional responsiveness not reliant on self - report would be to rate affect in response to emotional video clips , or affect during role - play scenarios . in addition , behavioral sampling using smartphone or text technology could be used to capture what a person is feeling at multiple times during the day . the use of new technologies provides great opportunity to bridge some of the shortcomings in the assessment of functioning , more generally , and the assessment of real - world functioning , specifically . remote data collection and remote reporting technologies include the use of portable electronic devices , such as tablet computers and smartphones , that provide opportunity for realtime assessment of day - to - day functioning . a major advantage of utilizing this technology is that it can enable the means to collect data on functioning over a period of days or weeks . this offers the potential to greatly improve measurement reliability while reducing resource expenditures in both cost and personnel hours , which have previously impeded the use of comprehensive functional assessments . applied to clinical trials , this would allow for collection of real - world functional data over the course of the study that could include a well - established baseline before treatment and continuous collection of functional data over the course of treatment . the amount of data collected on functioning may help to significantly enhance sensitivity for detecting change in functioning , which is a particularly important consideration for clinical trials . exploiting these technologies to good effect requires efforts to develop assessment applications and psychometric evaluation of these applications .

serious mental illness ( smi ) results in functional disability that imposes a significant burden on individuals , caregivers , and society . development of novel treatments is under way in an effort to improve the illness domains of cognitive impairment and negative symptoms and subsequently to improve functional outcomes . the assessment of functional outcomes in smi faces a number of challenges , including the proliferation of assessment instruments and the differential prioritization of functional goals among stakeholder groups . functional assessments relying on self- and informant report present a number of limitations . identifying alternative strategies to assess functioning that are reliable , valid , and sensitive to change is necessary for use in clinical trials . measures of functional capacity have been proposed for clinical trials investigating compounds to treat cognitive impairment in schizophrenia . alternative approaches employing effort - based decision making or daily activity recording using instruments such as the daily activity report may be more appropriate for studies focused on improving negative symptoms .

Introduction What is functional outcome and when should it change? Who reports about functional outcome? Does selection of functional outcome measure depend on the primary study target? The development and refinement of functional capacity measures What about studies of compounds designed to improve negative symptoms? How can new technologies improve functional assessment?

PMC4860798

the full oxidation of one glucose molecule ( oxidative phosphorylation ) within a cell in the presence of oxygen produces 38 molecules of adenosine - three - phosphate ( atp ) , which in turn represents the essential cellular fuel ( figure 1.a ) . the first step ( glycolysis , occurring in cytoplasm ) of glucose cellular respiration produces only 2 atp molecules and ends up with production of two molecules of pyruvic acid . if a given cell has access to oxygen , the pyruvic acid will be converted to acetyl - coenzyme a , which enters the krebs cycle ( citric acid cycle , occurring within mitochondria ) followed by the electron transport chain process ( occurring on the inner mitochondrial membrane ) that creates most of the atp molecules . it is the very last step within electron transport chain that needs oxygen to collect the terminal electron from the last cytochrome ( cyt a3 ) and become a nascent o to pick up 2 h and create one of the byproducts of aerobic cellular respiration - a water molecule . on the other hand , if the cells reside under hypoxic conditions , the pyruvic acid is not converted into acetyl - coenzyme a , but into a lactic acid - the process termed anaerobic cellular respiration ( lactic acid cycle ) . in the latter case , the net energy balance is only two atp molecules making the anaerobic glucose metabolism energetically highly inefficient process . while the lack of oxygen at the cellular level can occur at times of excessive physical activity ( resulting in subsequent muscle pain ) , it also represents a hallmark of highly invasive and fast - growing cancers [ ] . as the cancer cells multiply , their fast multiplication rate outgrows the angiogenesis so much so that while the glucose access to fast - growing cells could be sufficient the lack of blood vessels disrupts the level of oxygen needed for the full glucose oxidation . such a microscopic picture defines one of the fundamental properties of the cancer microenvironment that forces malignant cells to metabolize glucose through the lactic acid cycle . looking at chemical equations between aerobic and anaerobic glucose metabolism ( figure 1 ) one may conclude that cancer cells ( as well as healthy ones ) under hypoxic condition would need 19 times higher uptake of glucose to maintain the same metabolic level as well - oxygenated cells . this would , in turn , mean that the appropriate quantitative analysis of fdg - based pet images could help in pinpointing hypoxic segments ( by abundance only [ ] ) of the tumor by solely looking into very high uptake values , which would eventually be some 20 times greater than in well - oxygenated cancer cells . however , the isolation of the hypoxic target volumes is far from being that simple . apart from the fact that acute hypoxia in tumors develop as soon as one moves few hundred microns from the blood vessels , yet another important fact prevents fdg being an ideal hypoxia marker - the warburg effect . recently , interest in tumor metabolism has been revived partly as a result of the widespread clinical application of pet using fdg . fdg - based pet imaging has confirmed that most primary and metastatic cancers show a significant increase in the glucose uptake when compared to normal tissues . glycolysis involves the conversion of glucose to pyruvate and then to lactic acid , the waste product . in non - cancerous cells , mitochondria oxidize pyruvate to carbon dioxide and water in the presence of oxygen ( figure 2.a ) , and the glycolytic reaction is inhibited ( pasteur effect [ ] ) . conversion of glucose to lactic acid , even in the presence of oxygen is known as aerobic glycolysis ( figure 2.b ) or the warburg effect [ , ] . in one of his seminal papers [ ] the second phase of cancer formation represents a long struggle for existence by the injured cells to maintain their structure , in which a part of the cells die from lack of energy while another part succeeds in replacing the irretrievably lost respiration energy by fermentation energy ( from lactic acid cycle ) . warburg 's initial hypothesis that cancer results from impaired mitochondrial metabolism has been shown to be incorrect , but the observation of augmented glycolysis in tumors , even in the presence of oxygen , has been continually proven [ ] . while cancer cells do carry oxidative phosphorylation , the majority of glucose molecules taken by cancer cells ( 66% ) are metabolized through fermentation [ ] , a process that is ten times faster than full glucose oxidation . in addition to being energetically highly inefficient process glycolysis ( either anaerobic or aerobic ) , with its metabolic products ( such as hydrogen ions ) , cause constant acidification of the extracellular space , which might result in increased local toxicity [ , ] . nevertheless , despite these drawbacks , cancer cells consistently progress towards the wasteful and potentially toxic glycolytic phenotype . gatenby and gillies [ ] proposed that the consistent expression of up - regulated glycolysis is not accidental but represents a solution to the environmental growth constraints during tumor development . they suggest that increased glycolysis is an essential component of the malignant phenotype and , therefore , a hallmark of invasive cancers . transport enzymes of the glut and hexokinase families are up - regulated in tumor cells expressing the glycolytic phenotype , and the level of glut-1 glucose transporter expression has been shown to correlate with [ 18f ] fdg uptake in non - small cell lung cancer , for example [ ] . gatenby and gillies 11 describe the concept of carcinogenesis as a process that occurs by cellular evolution implying that common characteristics of malignant phenotypes , such as upregulation of glycolysis , are the result of active selection processes . they further argue that upregulation of glycolysis is likely to be an adjustment to hypoxia developing as pre - malignant tissue grows gradually further away from their blood supply . also , an augmented acid production from glycolysis upregulation leads to microenvironmental acidosis and requires further adjustments through somatic evolution to phenotypes resistant to acid - induced toxicity . finally , they conclude that cell populations that emerge from this evolutionary sequence have a compelling growth advantage , as they alter their environment through increased glycolysis in a way that is toxic to other phenotypes , but harmless to themselves . to further support the attempt by gatenby and gillies 11 in explaining the cause of warburg effect in aggressive tumors as a response to harsh environmental conditions know a priori they will encounter severe conditions in the future . consequently , they decide to switch their glucose metabolism to highly inefficient but the only possible ( and highly toxic ) metabolic pathway . to make their explanation more sounded , gatenby and gillies 11 speculate that : intuitively , it would seem that the darwinian forces prevailing during the somatic evolution of invasive cancers would select against a metabolic phenotype that is more than an order of magnitude less efficient than its competitors and that is environmentally poisonous . in other words , the accepted tenet of survival of the fittest would seem to generally favour populations with more efficient and sophisticated substrate metabolism . consequently , they suggest that glycolytic phenotype in cancers is directly governed by the evolutive mechanisms , over the relatively short time frame during which tumors develop . figure 3 illustrates the very point within a cellular glucose metabolism where the fate of the pyruvic acid is decided . in the presence of oxygen , the pyruvic acid will be converted into acetyl group and attached to coenzyme a , a process mediated by the so - called pyruvate dehydrogenase complex ( pdc ) . the complex consists of three enzymes : pyruvate dehydrogenase , dihydrolipoyl transacetylase , and dihydrolipoyl dehydrogenase . on the other hand , conversion of pyruvic acid to lactic acid requires one enzyme only - lactate dehydrogenase ( ldha ) . from the cellular kinetics point of view , one may ask a simple question : what is the probability that something goes wrong with either of the two possible metabolic pathways depicted in figure 3 ? synthesis of three enzymes , needed to provide conversion of pyruvic acid to acetyl coenzyme a , even in the presence of oxygen will be ( notably three times ) more prone to the errors than a transcription of only one ( competing ) enzyme . it was reported that many human cancers have higher ldha levels than normal tissues [ ] , but the correlation between oncogenes and glycolysis was poorly understood . the question could be then raised whether the warburg effect is only a consequence , or could it be at the root of the very cause of carcinogenesis ? a new hypothesis could be staged that the switch from aerobic cellular respiration to aerobic glycolysis leads to carcinogenesis , and the cell begins to develop the cancerous phenotype [ , ] at the point where the fate of pyruvic acid is decided . such a switch could be governed by the lack of complete pdc ( at least one of the enzymes is missing ) . while at this very moment the question whether warburg effect is a hen , or an egg remains at the level of pure hypothesis , several clinical observations might be called upon to support such proposition indirectly . while there is no apparent relation between type 1 diabetes mellitus patients and incidence of cancer to this date [ ] , several publications argue that there could be a lower cancer rate in patients with insulin - dependent diabetes . in 2003 , zendehdel et al . [ ] published results on cancer incidence in patients with type 1 ( insulin - dependent ) diabetes mellitus on a cohort of 29 187 patients , followed over a period of 30 years , during which they observed 355 incidences of cancer . such a low frequency ( 1% over 30 years , or 0.04% per year ) appears negligible when compared to 1.66 million cases of new cancer cases per year in the us ( 0.52% per year [ ] ) . [ ] reported on the lower incidence of cancer death mortality in diabetic patients ( 6.7% , both type 1 and 2 ) when compared to non - diabetic patients ( 13.4% ) using a cohort of 39 811 patients with the end - stage renal disease . it might be argued that cells in diabetic patients ( generally deprived of normal glucose uptake due to lacking insulin ) become trained ( to use rhetoric by blagosklonny [ ] ) by the microenvironment and well prepared as soon as glucose becomes available . once the glucose is phosphorylated by hexokinase and enters the glucose oxidation process , the cell is prepared not to waste the opportunity and gets the maximum out of the relatively scars glucose supplies . one could further argue that diabetic patient cells are making sure that the synthesis of the pdc is up and running flawlessly , to avoid wasteful pathway of cellular glucose metabolism . on the other hand , despite a relatively small amount of data published it appears that the incidence of cancer is also correlated with the increased intake of carbohydrates [ , , ] . one may argue that normal cells , exposed to increased supply of glucose would quickly switch towards the energetically inefficient pathway ( lactic acid cycle ) of burning glucose even in the presence of oxygen ( warburg effect ) since the source of energy ( glucose - atp ) are virtually inexhaustible . in addition , the atp production via fermentation is much faster ( as mentioned above ) , albeit highly ineficient , when compared to full oxydation . it is also of note that contrary to type 1 ( insulin - dependent ) , patients with type 2 diabetes mellitus have higher probability for cancer incidence [ ] . yet another detail deserves attention : type 1 diabetes is commonly regarded as a juvenile - onset diabetes as it often begins in childhood while the type 2 diabetes was considered an adult - onset diabetes . however , type 2 diabetes is becoming increasingly common in children [ ] who are more obese or overweight that could be correlated with carbohydrates rich diets . finally , the possible triggering of carcinogenesis by aerobic glycolysis , accompanied by increased glucose uptake , can be further supported by studies demonstrating increased glucose uptake observed to coincide with the transition from premalignant lesions to invasive cancer [ , ] . unlike warburg 's initial hypothesis that cancer cells metabolize glucose through aerobic glycolysis due to impaired mitochondrial function a new hypothesis was presented that the normal cell becomes cancerous at the point when it switches its glucose metabolism from oxidative phosphorylation to aerobic glycolysis . the new hypothesis that warburg effect corresponds to the very beginning of carcinogenesis such failure could be mediated by one ( or multiple ) of the well - known carcinogenic factors in synergy with an excessive supply of glucose in carbohydrates rich diets . the new hypothesis revolves around a point within cellular glucose oxidation at which the fate of pyruvic acid is decided . several observations have been presented to support such hypothesis : lower incidence of cancer in insulin - dependent type 1 diabetes mellitus patients ; increased cancer incidence in societies consuming high quantities of carbohydrates ; and the increased probability of synthesis failure of the pyruvate dehydrogenase complex consisting of three enzymes when compared to the synthesis of a single lactate dehydrogenase enzyme . contemporary agreement in explaining why tumor cells opt for aerobic glycolysis ( warburg effect ) that is far less efficient than oxidative phosphorylation at producing atp , is that it represents evolutionary adaptation to harsh microenvironmental conditions by using the carbon chains ( from the lactic acid ) as building blocks for synthesis of biomolecules ( nucleic acids , proteins , and lipids ) , which are essential for cell proliferation . however , even the acceptance of the aerobic glycolysis being a more adequate glucose metabolism pathway for cancer cells , the question of a hen or an egg remains : is the warburg effect just a consequence , or could it be the very cause of carcinogenesis ( figure 4 ) ?

ever since its discovery ( 1924 ) the warburg effect ( aerobic glycolysis ) remains an unresolved puzzle : why the aggressive cancer cells prefer to use the energetically highly inefficient method of burning the glucose at the cellular level ? while in the course of the last 90 years several hypotheses have been suggested , to this date there is no clear explanation of this rather unusual effect . even though it is commonly assumed that warburg effect is a consequence of carcinogenesis , yet another hypothesis could be brought up that the cellular switch to aerobic glycolysis may represent the very point in time when a normal cell becomes cancerous . furthermore , this switch may happen at the point where the fate of pyruvic acid is determined , caused by the inadequate supply of enzymes that promote citric as opposed to lactic acid cycle . currently , few clinical observations , like low cancer incidence in type 1 diabetes mellitus and increased cancer incidence in people on high carbohydrate diets might be called upon to support such hypothesis .

Economy of cellular energy balance Warburg effect, or aerobic glycolysis - hallmark of invasive cancers Contemporary explanation of the Warburg effect A new hypothesis Conclusion

PMC3665021

resistance training is reported to have many advantages such as weight control , prevention of osteoporosis , improvement of cardiovascular risk factors , and prevention of injury . free radicals , which are atoms and molecules that have an unpaired electron , can damage molecules that are important for cellular function , leading to a total loss of cellular function . free radicals attack the cells of critical ingredients such as lipids , proteins , and dna . results in the formation of superoxide radicals , 1 to 3 percent oxygen is converted to reactive hydroxyl radicals . the result , oxygen consumption increased electron transport through the respiratory chain and ultimately increases the production of free radicals . antioxidant systems of the human body through the production and use of antioxidants , disconnect the chain reaction caused by free radicals . the antioxidants react with free radicals ; free radicals are converted weak and have no desire to react with surrounding molecules . endurance athletes due to special circumstances , require an antioxidant system efficient than others ; the system maintains normal function of the body and adjust to increased oxidative stress induced by free radicals . one , physiological endogenous antioxidant systems , including glutathione peroxidase , catalase , and superoxide dismutase enzymes , and another defense against oxidative damage , antioxidants are derived from food sources . it is very important to have a proper diet for athletes to improve athletic performance of a certain antioxidant systems after intense exercise , although athletes may be unable to provide adequate antioxidant substances needed by the body from the diet . the best recognized ones are vitamins c and e antioxidants , among that are used mainly for sports supplements . vitamin c effectively reduces free radicals and also helps to restore the vitamin e. this vitamin also helps with iron absorption and has an important role in the formation of creatine , which is the main source of muscle energy . vitamins e and c have a role in strengthening and increasing with endogenous antioxidant glutathione and lipoic acid ; these two substances have a major role in the reconstruction of important vitamins from their radical forms . some studies have been conducted on exercise performance using antioxidant supplements known to improve performance but some of them do n't show any effect of antioxidant supplements on athletic performance and even expressed delayed recovery damaged muscle with the supplements , but antioxidant supplement still may be effective on exercise performance . further research is needed to answer whether supplements are helpful for athletes , especially female athletes , and limited studies have been done in this field . therefore , this study is aimed to investigate the effects of vitamin c and e supplementation on muscle damage , performance , and body composition in athlete women . sixty - four trained ( the best or most skilled members who had been doing aerobics for at least 3 years ) female athletes with the inclusion criteria were randomly recruited in isfahan sports club . this study was registered in the iranian registry of clinical trials ( www.irct.ir ) on october 7 , 2012 with irct registration number : irct201111095062n2 . considering mda as the main variable in the study sample and at the significance level of 5% and statistical power of 80% , to detect a measurable amount of 1.2 , 12 subjects were assigned to each of the groups . subjects were randomized using permuted of size4 blocks by another person to one of four groups ; a : vitamin c ( 250 mg / day ) , b : vitamin e ( 400 iu ) , c : vitamin c + vitamin e , and control ( placebo ) . they were initially willing to participate in the study and already had 3 to 6 years of experience in sports , were not taking antioxidant supplements in the previous month , were in complete physical health , and were free of disorders related to pregnancy and lactation . during the study , one participant was excluded from the sample due to a fracture in the leg . moreover , one participant due to digestive problems and weight changes and four participants withdrew from groups due to personal reasons . after explaining the purpose and details of the sample , written informed consent was obtained from each subject . each participant 's weight and height were measured and recorded using medical scales seca ( made in germany ) with an accuracy of 100 grams and stadiometer with an accuracy of 0.5 cm . athletes consumed a vitamin pill daily with their meals ; vitamin c ( iran , osveh ) or vitamin e ( iran , zahravi ) . vitamins placebo were prepared by the same company . in the beginning , each participant took supplements for four weeks and by counting the total number of pills remaining in the study , it was figured out if the participants followed the research code or not consumed . they were asked to keep doing their daily routine physical activity without changing it using a two - day food record . the information about their diet was collected at the outset and in the last week of intervention and analyzed with nutritionist software , version 4 . physical activity was assessed using a physical activity questionnaire and described according to metabolic equivalent - minutes a week standard . fat were also measured by skinfold caliper ( johnson , england ) at four points ; triceps , biceps , supra iliac , and subscapular . on the other hand , blood biochemical evaluation was performed at the baseline and at the end of the fourth week before giving supplements to participants . blood samples of 10 ml were taken from the brachial vein . to separate serum and blood samples , the samples were stored at room temperature for 1hour and then centrifuged at 2000 rpm for 15 minutes . myoglobin was measured by human myoglobin ( myo ) elisa kit ( co.glory science.analyses were performed with spss software , version 16 statistical package . the results are presented as mean standard error . due to the lack of normal distribution of variables , they were log transformed for normalization . method of multivariate analysis of variance was used to examine differences ( percentage changes before and after the intervention ) of variables among groups . paired t - test was used to compare changes within groups of variables and bonferroni test was used to compare changes between groups as a method of post - hoc comparisons of them . the age of the participants of the study was significantly different among the groups ( p < 0.05 ) but bmi and physical activity were not between groups [ table 1 ] . descriptive information compared the performance of sport , there was no significant difference between groups in vo2 max . also , vitamin supplements had no significant effect on subcutaneous fat between the groups , however , in the intergroup comparison , were significantly increased in group control ( p = 0.03 ) . but there was a significant increase in group 1(p= 0.04 ) [ table 2 ] . meanse : indicators of muscle damage , performance , and body composition compared between and within groups dietary intake was assessed using a two - day food records during software by nutritionist 4 that there was no significant difference between groups and within groups [ table 3 ] . the aim was to compare intake of vitamins a , e , c , d , -caroten , niacin , selenium , copper and zinc between the groups . the only significant difference was seen on selenium ( p < 0.05 ) , which had no effect on the other variables [ table 3 ] . the purpose of this study is to investigate the effects of vitamin c and e supplementation on muscle damage , performance , and body composition in athlete women . in a similar study , 2010 ) investigated the effects of combined vitamin c and e supplementation to healthy individuals on different measures of exercise performance after endurance training . the results suggested that administration of vitamins c and e to individuals with no previous vitamin deficiencies has no effect on physical adaptations to strenuous endurance training . dawson et al . ( 2002 ) investigated whether 4 weeks of daily supplementation with 500 or 1000 mg of vitamin c and 500 or 1000 iu of vitamin e could modify biochemical and ultrastructural indices of muscle damage following a 21 km run in 15 experienced males . they revealed that vitamin c and e supplementation ( 500 or 1000 mg or iu per day ) for four weeks does not reduce either biochemical or ultrastructural indices of muscle damage in experienced runners after a half marathon . ( 1998 ) examined whether a multivitamin and mineral supplement influenced the athletic performance of 30 competitive male athletes . at 0 , 3 , 6 , and 9 mo the runners performed a progressive treadmill test to volitional exhaustion for measurement of maximal oxygen consumption , peak running speed , blood lactate turn point , and peak post exercise blood lactate level . they showed that 3 month of multivitamin and mineral supplementation was without any measurable ergogenic effect . telford et al . examined the effect of vitamin and mineral supplementation over 7 to 8 months of training and competition in 82 athletes from four sports : basketball , gymnastics , rowing , and swimming . all athletes were monitored to ensure that the recommended daily intakes ( rdi ) of vitamins and minerals were provided by diet alone . sport - specific and some common tests of strength as well as aerobic and anaerobic fitness were performed . coaches ' assessment of improvement was also obtained . in general , this study provided little evidence of any effect of supplementation to athletic performance for athletes consuming the dietary rdis . singh et al . ( 1992 ) investigated the effects on physical performance of 90 d of supplementation with a high potency multivitamin - mineral supplement in 22 healthy , physically active men . they randomly assigned subjects to a supplement or placebo group ; both groups had similar physical characteristics . the results suggested that supplementation did not affect physical performance in well - nourished men who maintained their physical activity . ( 2007 ) examined the effects of antioxidant diet supplements on blood lactate concentration and on the aerobic and anaerobic thresholds and their adaptations to training were analyzed in 15 amateur male athletes . in the study , before and after the antioxidant supplements , the sportsmen performed a maximal exercise test on a cycle ergometer and maximal and submaximal physiological parameters were assessed together with blood lactate concentration . they revealed that antioxidant diet supplements induced lower increases in blood lactate concentration after a maximal exercise test and could improve the efficiency in which aerobic energy is obtained . macrae et al . ( 2006 ) investigated whether 6 wk of antioxidant supplementation ( as ) would enhance 30 km time trial ( tt ) cycling performance in eleven elite male cyclists . the study to test the effects of twice daily as containing essential vitamins plus quercetin ( frs ) and as minus quercetin ( frs - q ) versus a baseline tt ( b ) . thus , frs supplementation significantly improved high - intensity cycling tt performance through enhancement of power output . ( 2008 ) examined the effects of an antioxidant , coenzyme q10 ( coq10 ) , on muscular injury and oxidative stress during exercise training in 18 male students . they showed that indicate that coq10 supplementation reduced exercise - induced muscular injury in athletes . ( 2003 ) investigated the effect of antioxidant vitamins ( vitamin e and vitamin c ) on the exercise performance of rats . fifty male sd rats were randomly divided into control group ( c ) , exhausting exercise control group ( e ) , vitamin e group ( m1 ) , vitamin c group ( m2 ) , and vitamin e plus vitamin c group ( m3 ) . the rats in the exercising groups ( e , m1 , m2 , m3 ) were propelled for repeated exhausting runs on the treadmill for 4 weeks . they revealed that vitamin e can protect the mitochondria in the skeletal muscles and improve the exercise performance of rats , the effect of which can be enhanced by vitamin c , but vitamin c alone can not sufficiently achieve the effects . in the present study , there were no significant changes in the performance of athletes ; this could be due to different types of exercise and antioxidant supplementation or supplementation time or gender difference . the majority of studies in this area have been carried out on male athletes than female . also , a study conducted in china , and the result was an improved performance consumption of vitamin supplements , and a design very similar to this study , has been done on laboratory animals . this study could also be designed to examine the separate vitamins e , c and combined model of these vitamins and with the same study population , female athletes . however , there was a limit in the number of samples in this study , since more samples would yield more reliable results so this point is suggested to be considered in further related researches . future studies are suggested to be conducted with larger sample size and on other athletes using different antioxidant supplements . generally speaking , the results of this study showed that supplementation with vitamin c and e supplementation in groups had no significant effect on any of the parameters studied .

background : due to the special training conditions and lifestyle athletes require an antioxidant system that is more efficient than others . to keep this system optimal , many of them use antioxidant supplements . this study aimed to investigate the effects of vitamins c and e supplementation on muscle damage , performance , and body composition in athlete women.methods:the study was a 4-week randomized , double - blind clinical trial conducted on 64 trained female athletes recruited in isfahan sports club . they were randomly assigned to one of the following four groups ; a : vitamin c ( 250 mg / day ) , b : vitamin e ( 400 iu ) , c : vitamin c + vitamin e and control ( placebo ) . harvard step test was used to measure maximal oxygen consumption for performance , body composition , and damage marker ( myoglobin ) were measured before and after the intervention.results:comparing the result of the test in performance of sport , there was no significant difference between groups in vo2 max . also , vitamin supplements had no significant effect on subcutaneous fat between the groups , however , in the intergroup comparison , were significantly increased in group control ( p = 0.03 ) . but , there were no significant differences , change in myoglobin between the groups . there was a significant increase in group a ( p = 0.04).conclusions : vitamins c and e supplementation had no significant effect on any of the studied parameters .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION

PMC3943228

male labrador retrievers ( n=3 ) in our breeding colony were used in this study . five hundred microliters of ejaculate was placed into an eppendorf tube ( 1.7 ml , 17401 , sorenson bioscience , salt lake city , ut , usa ) , and then centrifuged for 5 min at 3,000 rpm . the supernatant was removed , and 1 ml of 0.01 m pbs ( phosphate buffered saline ; 0.35 g / l nah2po4 , 1.28 g / l na2hpo4 , 8.00 g / l nacl , wako , tokyo , japan ) was added . the suspension obtained was thoroughly mixed using a pipette and subsequently centrifuged for 5 min at 3,000 rpm . the supernatant was removed , and the pellet was resuspended with 1 ml of 0.01 m pbs . the washing procedures were repeated twice . the sperm suspension with 200 l of 0.01 m pbs was fixed by addition of 400 l of 3:1 methanol : acetic acid fixative and left at room temperature for 10 min . then , the tube containing sperm suspension was filled with the fixative , and then centrifuged for 5 min at 3,000 rpm . the sperm concentration was adjusted to 5 10 cells / ml , and 10 l of the suspension was then placed on a defatted glass slide . the concentration of sperm on the slide was examined using a light microscope ( alphaphot-2 ys2 , nikon , tokyo , japan ) . when the sperm density was unsatisfactory or the sample was contaminated with dust , the suspension was diluted , concentrated or washed with the fixative and centrifuged to get an appropriate concentration . preparations were dehydrated on a hot plate at 70 c for 60 min ( hardening ) . for decondensation of sperm nuclei , the specimen was treated with 1 m naoh for 3 , 4 or 5 min at room temperature , rinsed with pbs for 2 min , and then washed with distilled water several times . the specimen was dehydrated by running it through an ethanol concentration series ( 70% for 5 min and 100% for 30 min ) . after air - drying , the specimen was placed on a hot plate at 70 c for 60 min . for a single staining of the y - chromosome of canine sperm , 5 l of y chromosome probe labeled with digoxigenin and salmon sperm dna , canine suppression dna , and 50% formamide/10% dextran sulfate/2 x ssc mixture ( doy-10 , chromosome science laboratory , sapporo , japan ) was added to the specimen , and then covered with a cover glass . the preparation was transferred onto a hot plate set at 70 c for 5 min . the edges of the preparation were sealed with rubber cement ( sagisaka , tokyo , japan ) . thereafter , the cover glass was removed , and the specimen was washed several times with 50% formamide in 2xssc at 37 c for 20 min and with 1xssc alone at room temperature for 15 min . hybridization of the digoxigenin - labelled y probe was visualized with anti - digoxigenin cy3 ( chromosome science laboratory ) . the slides were counterstained with dapi ( 4 ' , 6-diamino-2-phenylindole , dihydrochloride , d1306 , invitrogen , carlsbad , ca , usa ) and mounted with antifade solution ( afm-01 , chromosome science laboratory ) . for double staining of x and y chromosome in canine spermatozoa , sex chromosome probes x - labelled with spectrumgreen and y - labelled with cy3 ( dxy-10 , chromosome science laboratory ) were used . the decondensation condition of sperm nuclei was selected according to the results of a single staining experiment . sperm samples were analyzed using a fluorescence microscope ( eclipse 80i , nikon ) equipped with dapi ( uv-2a , excitation 380420 nm , absorption 450 nm ) , spectrumgreen ( b2-a , excitation 450490 nm , absorption 520 nm ) and cy3 ( g-2a , excitation 510560 nm , absorption 590 nm ) filters ( nikon ) . digital images were acquired by a peltier - cooled ccd camera ( ds-5mc - l1 , nikon ) using a software for digital photo editing ( paint.net v3.5.10 ) . fish signals were analyzed visually using an ocular grid by counting random microscope fields and scoring 500600 spermatozoa per sample . spermatozoa were scored only if they were intact and non - overlapped , had a clearly defined border and had not decondensed to more than twice the size of a non - decondensed sperm head , which could produce large and sometimes fragmented fish signals . semen samples were collected as described above , and seminal plasma was removed by centrifugation at 2000 rpm for 15 min . the sorted spermatozoa were obtained following the general procedure described by schenk et al . briefly , spermatozoa were diluted with talp at a sperm concentration of 2 10 cells / ml , and then stained with hoechst 33342 ( 81.0113.4 m , h3570 , invitrogen ) and incubated for 3540 min at 35 c. an equal volume of talp containing 2.67% of purified egg yolk and 0.002% of food red ( fd & c # 40 , cas 25856 - 17 - 6 , invitrogen ) was added to the sperm suspension . the stained spermatozoa were sorted with a flow cytometer ( moflo - sx , dakocytomation , fort collins , co , usa ) operating at 40 psi and with a laser power of 175 mw . all experiments were carried out in accordance with the guidelines for the care and use of animals approved by obihiro university of agriculture and veterinary medicine . a chi - squared test was used to investigate the deviation from the expected ratio of 50:50 ( x : y ) . male labrador retrievers ( n=3 ) in our breeding colony were used in this study . five hundred microliters of ejaculate was placed into an eppendorf tube ( 1.7 ml , 17401 , sorenson bioscience , salt lake city , ut , usa ) , and then centrifuged for 5 min at 3,000 rpm . the supernatant was removed , and 1 ml of 0.01 m pbs ( phosphate buffered saline ; 0.35 g / l nah2po4 , 1.28 g / l na2hpo4 , 8.00 g / l nacl , wako , tokyo , japan ) was added . the suspension obtained was thoroughly mixed using a pipette and subsequently centrifuged for 5 min at 3,000 rpm . the supernatant was removed , and the pellet was resuspended with 1 ml of 0.01 m pbs . the washing procedures were repeated twice . the sperm suspension with 200 l of 0.01 m pbs was fixed by addition of 400 l of 3:1 methanol : acetic acid fixative and left at room temperature for 10 min . then , the tube containing sperm suspension was filled with the fixative , and then centrifuged for 5 min at 3,000 rpm . the sperm concentration was adjusted to 5 10 cells / ml , and 10 l of the suspension was then placed on a defatted glass slide . the concentration of sperm on the slide was examined using a light microscope ( alphaphot-2 ys2 , nikon , tokyo , japan ) . when the sperm density was unsatisfactory or the sample was contaminated with dust , the suspension was diluted , concentrated or washed with the fixative and centrifuged to get an appropriate concentration . preparations were dehydrated on a hot plate at 70 c for 60 min ( hardening ) . for decondensation of sperm nuclei , the specimen was treated with 1 m naoh for 3 , 4 or 5 min at room temperature , rinsed with pbs for 2 min , and then washed with distilled water several times . the specimen was dehydrated by running it through an ethanol concentration series ( 70% for 5 min and 100% for 30 min ) . after air - drying , the specimen was placed on a hot plate at 70 c for 60 min . for a single staining of the y - chromosome of canine sperm , 5 l of y chromosome probe labeled with digoxigenin and salmon sperm dna , canine suppression dna , and 50% formamide/10% dextran sulfate/2 x ssc mixture ( doy-10 , chromosome science laboratory , sapporo , japan ) was added to the specimen , and then covered with a cover glass . the preparation was transferred onto a hot plate set at 70 c for 5 min . the edges of the preparation were sealed with rubber cement ( sagisaka , tokyo , japan ) . thereafter , the cover glass was removed , and the specimen was washed several times with 50% formamide in 2xssc at 37 c for 20 min and with 1xssc alone at room temperature for 15 min . hybridization of the digoxigenin - labelled y probe was visualized with anti - digoxigenin cy3 ( chromosome science laboratory ) . the slides were counterstained with dapi ( 4 ' , 6-diamino-2-phenylindole , dihydrochloride , d1306 , invitrogen , carlsbad , ca , usa ) and mounted with antifade solution ( afm-01 , chromosome science laboratory ) . for double staining of x and y chromosome in canine spermatozoa , sex chromosome probes x - labelled with spectrumgreen and y - labelled with cy3 ( dxy-10 , chromosome science laboratory ) were used . the decondensation condition of sperm nuclei was selected according to the results of a single staining experiment . sperm samples were analyzed using a fluorescence microscope ( eclipse 80i , nikon ) equipped with dapi ( uv-2a , excitation 380420 nm , absorption 450 nm ) , spectrumgreen ( b2-a , excitation 450490 nm , absorption 520 nm ) and cy3 ( g-2a , excitation 510560 nm , absorption 590 nm ) filters ( nikon ) . digital images were acquired by a peltier - cooled ccd camera ( ds-5mc - l1 , nikon ) using a software for digital photo editing ( paint.net v3.5.10 ) . fish signals were analyzed visually using an ocular grid by counting random microscope fields and scoring 500600 spermatozoa per sample . spermatozoa were scored only if they were intact and non - overlapped , had a clearly defined border and had not decondensed to more than twice the size of a non - decondensed sperm head , which could produce large and sometimes fragmented fish signals . semen samples were collected as described above , and seminal plasma was removed by centrifugation at 2000 rpm for 15 min . the sorted spermatozoa were obtained following the general procedure described by schenk et al . briefly , spermatozoa were diluted with talp at a sperm concentration of 2 10 cells / ml , and then stained with hoechst 33342 ( 81.0113.4 m , h3570 , invitrogen ) and incubated for 3540 min at 35 c. an equal volume of talp containing 2.67% of purified egg yolk and 0.002% of food red ( fd & c # 40 , cas 25856 - 17 - 6 , invitrogen ) was added to the sperm suspension . the stained spermatozoa were sorted with a flow cytometer ( moflo - sx , dakocytomation , fort collins , co , usa ) operating at 40 psi and with a laser power of 175 mw . control samples consisted of unsorted spermatozoa from the same dogs . all experiments were carried out in accordance with the guidelines for the care and use of animals approved by obihiro university of agriculture and veterinary medicine . a chi - squared test was used to investigate the deviation from the expected ratio of 50:50 ( x : y ) .

abstract effective preselection of sex has been accomplished in several species of livestock and also in humans using the flow cytometric sperm sorting method . a guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing . the standard validation method is flow cytometric remeasurement of the dna content of the sexed sperm . since this method relies on the same instrument that produced the original sperm separation , it is not truly independent . therefore , to be able to specifically produce either male or female offspring in the dog , we developed a method of direct visualization of sex chromosomes in a single sperm using fluorescence in situ hybridization ( fish ) as a validation method . denaturation of canine spermatozoa by immersion in 1 m naoh for 4 min yielded consistent hybridization results with over 97% hybridization efficiency and a good preservation of sperm morphology . there was no significant difference between the theoretical ratio ( 50:50 ) and the observed ratio of x- and y - chromosome - bearing spermatozoa in any of the three dogs . in addition , the mean purities of flow - sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for the x chromosome fraction and 89.6% for the y chromosome fraction . this sorting was evaluated by using the dual color fish protocol . therefore , our results demonstrated that the fish protocol worked reliably for both unsorted and sexed sperm samples .

Methods Animals and semen preparation Decondensation FISH Fluorescence microscopy and quantitative evaluation Flow cytometric sperm sorting Statistical analysis

PMC2904544

regular physical activity ( pa ) reduces the risk of chronic disease , including some types of cancer , diabetes , obesity and cardiovascular disease ( 1 ) . despite this evidence , pa levels continue to be far from ideal in most parts of the world ( 2 ) . of special concern is the evidence indicating that children , particularly in the developed world , establish a sedentary lifestyle early in life ( 3 ) . some developing countries are experiencing similar patterns associated with lifestyle changes , such as sedentary habits and high caloric intake that contribute in part to the worldwide obesity epidemic ( 4 ) . the latin american region is experiencing increasing rates of obesity among youth ( 59 ) . in developing countries , high prevalence of overweight and obesity lead to early mortality in adulthood ( 10 ) . children today have fewer opportunities to be active in a safe and independent manner , especially in large cities in developing countries that are rapidly urbanizing ( 11 ) . according to a birth cohort study conducted in pelotas , brazil , the prevalence of sedentary lifestyles , characterized by excessive tv viewing and lack of pa , among youth is as high as 58% ( 12 ) . factors that decrease energy expenditure , such as the declining time for physical education ( pe ) in schools , may play an important role in the prevalence of overweight among children . because students spend large amounts of time in school , there is a great potential for increasing their level of pa through school - based interventions ( 13,14 ) . in a systematic review of 14 evidence - based pa promotion strategies , the us guide to community preventive services ( community guide ) found strong supporting evidence that school - based pe programs increased pa among school children ( 15 ) . the school - based pe interventions included modified pe classes , generally with more classroom time and more moderate - to - vigorous pa ( 16 ) . the recommendations of the community guide and its process of systematic review have not yet been systematically examined and applied in developing countries . many promising pa interventions are being carried out in latin america ( 17 ) ; however , their effectiveness has generally not been evaluated . the guide for useful interventions for activity in latin america ( guia ) was initiated in 2005 to examine and promote evidence - based strategies , with the intention of increasing pa in latin america ( 18 ) . the purpose of the initial phase of guia was to conduct a review of the latin american literature on community - based pa promotion strategies and to determine the applicability of the community guide recommendations in this region . according to community guide criteria , school - based pe was found to be the only type of pa intervention with a strong enough body of evidence to make a practice recommendation ( 18 ) . this article focuses on the results of the guia systematic review related to school - based pe programs in latin america . the aims of the article are to ( i ) describe selected school - based pe interventions from the guia review ; ( ii ) discuss implications of effective school - based pe recommendations in the latin america region ; ( iii ) propose approaches for implementing these interventions in latin america ; ( iv ) identify gaps in the research literature related to pa promotion in latin american youth . although this article focuses specifically on the school - based pe findings of the guia literature review , the method used to search , categorize and abstract the papers for the present study were the same used in the overall review . therefore , in order to understand the process of reviewing and evaluating the latin america literature related to pa interventions , it is necessary to review the methods previously described ( 18 ) . first , five databases of peer - reviewed literature and brazilian theses were systematically searched for studies of pa community interventions by using search terms in portuguese , spanish and english . intervention studies were then synthesized in one - page summary tables , categorized by the 14 community guide pa intervention categories ( 16 ) and screened for inclusion in a full abstraction process . this process followed the community guide procedures ( 19 ) , with some modifications ( 18 ) . on the basis of previously described criteria ( 15 ) , studies were evaluated by two measures : design suitability ( classified as greatest , moderate and least [ appendix 1 ] ) and quality of intervention execution ( classified according to the number of limitations to assess the strength of the evidence [ appendix 2 ] ) . relative effect sizes for the school - based pe intervention studies were calculated as the net percentage change from baseline for all reported measures of aerobic capacity and physical activity levels and for all time periods . the formula for calculating the net effect ( figure 1 ) varied depending on the study design with or without a control group or pre - intervention outcome measurement ( 16 ) . to identify common characteristics of the school - based pe interventions , the authors reviewed each of the school - based pe articles and abstraction forms in detail , categorizing and listing intervention components . core components were determined according to frequency of use by most of the studies and also by taking into account the intervention activities recognized by the community guide ( 16 ) . additionally , brazilian experts provided input to address issues related to pe typically not available in the literature . these experts made available additional literature sources , such as peer - non - reviewed manuscripts and meeting reports , not included in the evidence - based review . the systematic literature review yielded five school - based pe intervention studies ( 2024 ) , out of six that met all of the inclusion criteria ( 18 ) . the excluded study was a duplicate of one included thesis ( 20 ) , which contained more details about the intervention and evaluation for the abstraction process than the peer - reviewed article . all five studies had sufficient quality of design , execution and detail regarding the intervention and outcomes to undergo the second phase of the abstraction process . three studies used randomized group design ( 20,21,23 ) and two used a non - randomized group design with a concurrent comparison group ( table 1 ) ( 22,24 ) . one study was conducted in elementary public schools in brazil ( children aged 710 years ) ( 20 ) , two were carried out in public schools in chile ( first- through ninth - grade classes ) ( 21,22 ) and the remaining two took place in us elementary schools mainly attended by latino students and located on the us / mexico border ( 23,24 ) . two studies ( 23,24 ) reported minutes spent in moderate and vigorous pa , and one study ( 20 ) reported four different outcomes related to behavior : percentage of children biking or walking to school , estimated energy expenditure based on observed pa behavior of children , percentage of children who were very active during pe classes and percentage of time spent walking during pe classes . further reported outcomes were aerobic capacity as estimated by maximal oxygen uptake ( vo2 max ) ( 21 ) , results from timed runs ( 23 ) or endurance testing ( shuttle runs ) ( 22 ) . although the outcomes , ascertainment methods , and duration of interventions varied between studies , there were consistent positive increases in pa levels for all outcomes measured during pe classes ( relative net effects ranged from 7% to 307% ) in all three randomized studies ( 20,21,23 ) ( figure 1 ) . the extreme magnitude of the 307% net effect is explained by the low baseline walking levels in the intervention group versus the control group ( 5.6 vs. 14.4% , respectively ) . at post - test the intervention group increased time spent walking during pe classes nearly 56 times that of baseline , while the control group increased walking time by only 1.5 times baseline . likewise , endurance ( 23 ) and active transportation to school ( 20 ) outcomes showed small but positive net effects for the same group of studies . with the exception of one cohort in heath ( 24 ) , the non - randomized studies increased moderate and vigorous pa levels during pe classes . these increases ranged from 5% to 69% for moderate and 50% to 55% for vigorous pa levels during pe classes . the results from the shuttle run test ( 22 ) demonstrated increased endurance performances among boys ( 35% ) and girls ( 37% ) . after reviewing the details of each study ( table 1 ) , four types of intervention activities were identified and summarized as the core components presented in table 2 . core components included the provision of information about the benefits and importance of health and active living ( 20,22 ) ; types of pa suitable for a particular age range ( 20 ) ; overall pe class planning ( lesson plan development , class and environment management ) and methods of pa practice ( 23,24 ) ; increases in the duration of the already scheduled pe classes ( 23,24 ) ; addition of extra pe classes per week ( 21,22 ) ; use of age - specific and enjoyable activities ( 2024 ) ( such as balance exercises , dancing , jumping , and running ) ; availability of new materials for classes ( balls , basketball boards ) ( 2022 ) ; and adaptable , flexible and culturally sensitive activities based on ethnicity or socioeconomic status ( 20,2224 ) . characteristics of the five school - based intervention abstracted studies core components summarized from the five intervention studies . our results identified several common characteristics , the core components ( table 2 ) , of successful school - based pe interventions in latin america that were comparable to some of those used in the community guide . these included the addition of supplementary pe classes , the lengthening of existing pe classes , and an increase in time spent in moderate and vigorous pa without necessarily changing duration of pe classes ( 16 ) . four ( 20,2224 ) out of the five studies reviewed in the current study were adaptations of north american school - based interventions and therefore delivered analogous activities to the 13 studies reviewed by the community guide , both in terms of content and quality . with appropriate adjustments for national , local , and cultural contexts , the identified core components from interventions from two schools in chile , a brazilian elementary school and two elementary schools on the us / mexican border could be a logical approach to guide pe recommendations and implementation across diverse countries , cultures and populations such as those found in latin america . high - quality school - based pe programs , combining all or some of the core components described here , increase pa among children in latin america in the same way as they do in the usa ( 18 ) . similar to the guia literature review ( 18 ) , the 13 us qualifying studies revealed consistent increases in time spent in pa at school ( 16 ) , the net increase in the amount of pe class time spent in moderate and vigorous pa was 50% , ranging from 6% to 125% . the percentage of class time increase in moderate and vigorous pa was 10% , ranging from 3% to 15% , although one study reported a 762% increase from a very small baseline value . two studies showed increases in energy expenditure , and 11 studies reported increases in aerobic capacity with a median of 8% ( interquartile range 3% to 19% ) ( 16 ) . when comparing intervention effects for the same outcomes , the five studies from the present study are generally consistent with the us cg results , both in direction and effect size . some of the us cg net effects also had a wide range , particularly for the amount and percentage of pe class time spent in moderate and vigorous pa . the three studies that reported increased levels of pa during pe class ( 20,23,24 ) used the same observational method ( 16 ) to measure the amount of pe class time spent in moderate and vigorous pa , but for different outcomes , such as the time spent being very active and the amount of walking during pe classes ( 20 ) . the wide range in net effect sizes reflects differences in the degree of change in the outcome measures for intervention and comparison ( if applicable ) groups before and after the intervention . they also reflect the magnitude of the pre - intervention measures ( see figure 1 note ) . insufficient evidence supported recommending classroom - based health education to increase pa , mainly because of an insufficient number of qualified studies ( n = 3 ) ( 18 ) . these interventions consist of multicomponent health education classes in elementary , middle , or high schools that aim to help students to develop the skills they need to make rational decisions about adopting healthier behaviors ( 16 ) . net percentage change in physical activity from baseline for the school - based physical education intervention studies source : adapted from kahn e , ramsey l , brownson r , et al . am j prev med . 2002 ; 22(4 ) : 73107 . among the three reviewed studies , the net effects for various outcomes ( e.g. frequency of exercise - related behaviors , any moderate and vigorous pa in the previous week ) ranged from 0.0 to 0.2 ; net effects for 11 of the 14 outcomes were between 0 and 0.1 . these net effects tended to be much lower in magnitude than those observed for the school - based pe interventions , which suggests that merely providing health information and promoting behavioral skills for healthy decision - making may be insufficient to significantly change pa behavior . however , more research of classroom - based health education , both in the usa ( 16 ) and in latin america , is needed to confirm this finding . the impact of an intervention is determined not only by its reach and efficacy but also by the extent to which the intervention is adopted , implemented as intended and maintained at the system and individual level ( 25 ) . an effective intervention needs to have broad reach and be feasible to implement in real - world settings in order to make a public health impact ( 25 ) . some of the major challenges for interventions are finding creative and cost - effective ways to implement evidence - based programs and policies at the population level , while providing adequate information and resources ( e.g. infrastructure , training , proper economic and social environments ) ( 26 ) . this is particularly important in a region where legislation to promote school - based pe is highly variable . in addition , it is key to consider differences in terms of social influences that may affect children 's and adolescents behavior ( e.g. family , social networks , culture ) when adapting programs that are effective in a specific setting or population ( 27 ) . although the purpose of school - based pe is larger than enhancing physical fitness only , it also has great potential for pa promotion because of its effectiveness in increasing pa among children ( 18 ) and because school programs reach virtually all children at relatively low cost with preexisting infrastructure . in a context of increased academic and political credibility for pa promotion and pe during the past decade ( 28 ) , supportive school pe policies appear to exist in many latin american countries ( 29,30 ) . in these countries , even though pe is mandatory for students in public and private elementary and secondary schools , the requirements concerning frequency and duration of classes is generally low ( 31 ) . an optimal pe curriculum should include a variety of learning and practice experiences that will provide the basis for active living in the adult years ( 32 ) . however , according to experts in the area , pe programs in latin america are often of low quality and quantity . therefore , a comprehensive school policy to increase pa practice during school - based pe and enhance its quality is required . in addition , frequency and duration of classes is often less than optimal because of challenges related to salary , work environment , physical space and curriculum structure ( 31 ) ; consequently , barriers to implementation exist primarily within the school systems and need to be addressed as well . according to melo ( 28 ) , there is a call for academic institutions where pe teachers are trained to push for an adequate transition from what is learned in the academic world to what is needed in everyday practice in the schools . often , the fields of academic knowledge production ( e.g. universities / research institutes ) and policy formulation / implementation are very different , frequently with incompatible goals and methods ( 33 ) . therefore , collaborative partnerships between researchers and practitioners , along with regulatory and legislative enforcement and political commitment and support for pe , are particularly desirable to help bridge the gap between theory and practice and for successful and sustainable program implementation the five identified studies applied diverse ways to measure similar or same outcomes ( e.g. moderate and vigorous pa levels during pe classes ) , which limited the ability of the previous review to derive a summary measure for the effect of school - based pe across the five reviewed studies . the net effect plot is an effort to be transparent in illustrating the variability in measures and lack of a gold standard in pa measurement in kids . by computing the net intervention effect although the literature search was conducted to identify interventions delivered in latin america , we discuss findings from only five studies , two of which were implemented on the us / mexican border ( 23,24 ) . however , because these interventions took place in schools attended mainly by hispanic children of low socioeconomic status , and since information on quality or availability of equipments and facilities was not available in the reviewed papers , we considered this population and setting comparable to those from other latin american countries . the three remaining studies were limited to chile ( 21,22 ) and brazil ( 20 ) . this limitation should be taken into account when disseminating school - based pe interventions in latin america . another consideration is the variability in the interventions duration ( 4 months to 3 years ) . the two longest studies ( 23,24 ) are funded adaptations of a broad , well - designed , tested and documented intervention study , which could justify in part the length of these two interventions . the three remaining studies ( 2022 ) were much less supported and were single initiatives largely developed by academic institutions ( university of chile , catholic university of chile and federal university of sao paulo ) in collaboration with local health or education sectors . nevertheless , the two longest interventions ( 23,24 ) did not result in larger net effect for similar outcomes when compared with the study with shortest duration ( 20 ) . identifying interventions to increase pa levels in school settings is particularly important in latin america because of the rapid demographic , epidemiologic and nutritional transitions associated with the increase in risk factors for chronic disease , including obesity and physical inactivity ( 4 ) . these problems are unequally distributed across populations , and they disproportionately affect the most impoverished latin americans . given the limited literature on evidence - based interventions for pa promotion in latin american countries , developing recommendations to address physical inactivity among youth is essential . the five studies in the guia review ( 18 ) and the 13 studies from the community guide review ( 16 ) were of sufficient quality to be included in the evidence - based review process and form the basis for the recommendation of school - based pe for pa promotion among children and adolescents in latin america . even though this may appear to be a small number of studies upon which to base global public health policy , relatively few other public health intervention recommendations are supported by systematic reviews of the scientific literature ( 27 ) . further implementation of school - based pe interventions could greatly benefit from systematic evaluation and publication of peer - reviewed studies on schools pe efforts to promote pa . given the importance of promoting pa in youth to improve the health of latin americans , our results provide the impetus to implement high - quality school - based pe interventions in this region . enhancing the quality of pe in the schools and increasing the level of pa among youth depends on capacity building and training for pe teachers and instructors , appropriate changes in the pe curricula , proper infrastructure including equipment and materials , and adjustments to various political , cultural and socioeconomic characteristics . additionally , future research is needed to identify other practical key elements , such as legislation , policy , barriers and facilitators for promoting school - based pe in latin america . the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention .

this article focuses on results of the systematic review from the guide for useful interventions for activity in latin america project related to school - based physical education ( pe ) programs in latin america . the aims of the article are to describe five school - based pe programs from latin america , discuss implications for effective school - based pe recommendations , propose approaches for implementing these interventions , and identify gaps in the research literature related to physical activity promotion in latin american youth . following the us community guide systematic review process , five school - based pe intervention studies with sufficient quality of design , execution and detail of intervention and outcomes were selected for full abstraction . one study was conducted in brazil , two studies were conducted in chile and two studies were conducted on the us / mexico border . while studies presented assorted outcomes , methods and duration of interventions , there were consistent positive increases in physical activity levels for all outcomes measured during pe classes , endurance and active transportation to school in all three randomized studies . except for one cohort from one study , the non - randomized studies showed positive intervention effects for moderate and vigorous physical activity levels during pe classes . the core elements of these five interventions included capacity building and staff training ( pe specialists and/or classroom teachers ) ; changes in the pe curricula ; provision of equipment and materials ; and adjustment of the interventions to specific target populations . in order to translate the strong evidence for school - based pe into practice , systematic attention to policy and implementation issues is required . ( global health promotion , 2010 ; 17(2 ) : pp . 0515 )

Introduction Methods Results Discussion Conclusions Disclaimer

PMC3418661

integral membrane proteins , particularly g - protein - coupled receptors ( gpcrs ) , are the biological targets for half of all the small molecule pharmaceuticals on the market today [ 13 ] . membrane transport proteins , such as p - glycoprotein and related efflux pumps , are thought to impart chemotherapy agent resistance by transporting the drugs from the cytoplasm faster than they can diffuse back , thus lowering the effective drug concentrations at the site of action . even the common cold ( rhinovirus ) invades the cell by first binding to specific cell surface proteins [ 57 ] , at least some of which are thought to involve glycosylated and sialylated extracelluar domain recognition sites [ 7 , 8 ] . clearly , integral membrane proteins play key biological roles in cell signaling , transport , and pathogen invasion . as such , membrane proteins also play key clinical roles in drug efficacy and resistance and should have a larger role in clinical diagnostics and personalized medicine . however , quantitative clinical assays ( e.g. , immunosorbent assays ) for this important class of proteins remain elusive and are generally limited to serum - soluble extracellular fragments . many serum markers for cancer detection and treatment monitoring such as ca-125 ( a serum - soluble fragment of mucin-16 approved for recurrence monitoring of ovarian cancer ) , ca 15 - 3 ( a serum - soluble fragment of mucin-1 approved for recurrence monitoring of breast cancer ) , svegfr ( a serum - soluble fragment of the vascular endothelial growth factor receptor that is implicated as a prognostic marker in lung cancer ) , and segfr ( a serum - soluble fragment of endothelial growth factor receptor that is implicated as a theranostic marker for trastuzumab treatment in breast cancer ) are currently only accessible for clinical assays once extracellular fragments are shed from the tumor cell membranes into the circulatory system . other membrane protein biomarkers such as her-2/neu ( an oncogenic growth factor receptor approved for use in herceptin therapy guidance ) and the estrogen receptor ( an indicator for hormonal therapy in breast cancer ) are currently only accessible through gene - based assays . yet , genetic assays are unable to detect potentially clinically relevant posttranslational modifications , such as glycosylation , phosphorylation , acetylation , ubiquitination , and editing . furthermore , as has been well established for more than a decade , measurements of mrna levels , which are produced transiently , do not correlate well to protein levels , which accumulate over time [ 13 , 14 ] . detergents also mediate membrane protein solubility in aqueous solutions , which is a prerequisite for further protein purification . the surfactant concentrations required to keep most membrane proteins in aqueous solution also typically denature immunoglobulins , precluding their use for immunoaffinity purification and enrichment . therefore , purification of membrane proteins is often very tedious and is made more so because surfactants can only partially mimic the lipid bilayer environment of the protein in nature . thus , many membrane proteins no longer retain their native biological conformations or activities in surfactant solutions , except in isolated cases . mitic et al . showed how the recovery of claudin-4 ( with four transmembrane sequences ) from insect cell cultures failed to consistently track total protein recovery over 37 different surfactants tested , ranging from 0 to 169% of the sodium dodecyl sulfate ( sds ) surfactants also create limitations on further proteomic analysis of membrane proteins , since subsequent polyacrylamide gel electrophoresis of the recovered proteins generally requires sds , or other ionic surfactants such as perfluorooctanoic acid . with the exception of newer acid - cleavable forms , surfactants can produce ionization problems for mass spectrometric analyses , except at very low concentrations [ 22 , 23 ] , which are too low to support solubility of membrane proteins . surfactants also bind to surfaces , significantly altering the behavior of liquid chromatographic media . because of the problems surfactants pose in chromatographic and mass spectrometric proteomic analyses , several membrane protein extraction schemes have been reported based on chaotropic agents and organic solvents . report a 4 : 1 : 1 mixture of ethanol : acetonitrile : water as being useful for recovering membrane proteins of mitochondrial and spherosomal origin in a system that is optically transparent in the range of 200700 nm . the lower end of this optical range is particularly useful for studying protein structure by circular dichroism or quantification by absorbance . report on the use of aqueous methanol , trifluoroethanol , and aqueous urea for the extraction of membrane proteins from red blood cells , finding that each solvent system liberated different membrane proteins . cordwell has advocated the use of a series of potent chaotropic agents and detergents ( increasing progressively in strength ) for protein extraction and fractionation , ending with thiourea and amidosulfobetaine-14 . he applied this method to gram - negative bacteria , cultured mammalian cells , and heart tissue . in all these cases , the more readily soluble proteins are generally recovered first from the patient sample in standard aqueous buffers from which they can be more readily purified and assayed since all the solvents , detergents , and chaotropic agents necessary to recover and solubilize the integral membrane proteins are incompatible with the downstream separation and purification schemes . therefore , the only applicable immunoaffinity technique that can be used on most membrane proteins after surfactant extraction is western blotting , which has only been sparing applied in clinical settings ( e.g. , early human immunodeficiency virus testing , early bovine spongiform encephalopathy testing , and lyme disease ) . even these were quickly replaced when more robust nucleic acid or enzyme - linked immunosorbent assay ( elisa ) alternatives became available . another classic proteomic approach to membrane protein analysis involves digestion of the generally soluble intra- and extracellular domains from the generally insoluble transmembrane regions . used this approach in combination with immobilized metal ion affinity chromatography ( imac ) to study the phosphopeptides resulting from trypsin treatment of the plasma membrane fraction isolated from cultured arabidopsis cells . however , membrane proteins can be refractory to digestion , particularly to trypsin , and nonspecific digestion enzymes ( e.g. , pepsin and elastase ) are more commonly employed for this purpose . the challenge , therefore , is to control the degree of digestion so that optimal recoveries are obtained . the resulting mixtures of partially digested peptides limit detection of the subsequent peptides by distributing the available signal over more distinct , but related , mass spectrometric species . furthermore , this approach is generally only suited for global proteomic analysis by liquid chromatography and mass spectrometry since the resultant peptides are often not immunologically active and mixtures of peptides are created from all the membrane proteins found in the sample ( both diagnostic and nondiagnostic ) . with the exception of qualitative immunohistochemistry , clinical exploitation of integral membrane proteins has heretofore been limited by our ability to recover these proteins in a form suitable for quantitative immunoassays and rapid proteomic characterization . in this paper , we describe a new approach for the combined quantitative recovery of both cytosolic and integral membrane proteins in a buffer system immediately suitable for direct use in immunosorbent assays and subsequent mass spectrometric analyses . this approach uses the commercial proteosolve - td1 and -td2 extraction buffers , developed in our laboratory and commercialized by pressure biosciences ( pbi ) , in combination with pbi 's pressure cycling technology ( pct ) . this new approach is demonstrated by extraction and analysis of several different biomarker proteins from fresh frozen human metastatic ovarian tumor tissues . fresh frozen samples of metastatic ovarian tumors ( surgically isolated from the omentum of several different individuals ) were purchased from bio - options ( fullerton , ca ) . all samples were reported to have been obtained from surgical resections of stages iii and iv ovarian cancer patients . the frozen tumor samples were finely diced and mixed prior to extraction to provide better sample homogeneity . diced tumor tissue ( 200 mg ) was weighed into an aluminum weigh boat , precooled over dry ice , to keep the samples frozen during processing . a mortar and pestle were precooled by addition of liquid n2 until a permanent lake of liquid n2 could be maintained in the mortar . the tissue sample was added to the lake of liquid n2 and cryogenically ground under liquid n2 to a fine powder ( about the consistency of corn starch ) . the liquid n2 was allowed to evaporate , and the frozen tissue along with any frozen condensate was transferred to a pulse tube ( an integral part of the barocycler device ) , which was precooled in a bed of dry ice . three different buffer systems were used to extract proteins from the ground tumor tissue samples . the control extraction buffer , adapted from song et al . for the homogenization of liver tissue for subsequent protein recovery and analysis , consisted of 20 mm hepes adjusted to ph 7.5 with naoh . this buffer also forms the basis of the proteosolve - td1 extraction buffer ( pressure biosciences , south easton , ma ) , which contains additional agents for membrane protein extraction and stabilization . the proteosolve - td2 extraction buffer ( pressure biosciences ) was subsequently formulated with additional agents to stabilize the ground tissue dispersion during barocycler operation , improving the reproducibility of protein extraction between samples . all these buffers were formulated to be compatible with subsequent protein labeling chemistries ( e.g. , aldehyde schiff - base , n - hydroxysuccinimide , or iodoacetamide ) to facilitate quantitative stable isotope work , such as isotope - coded affinity tags ( icats ) , isobaric tags for relative and absolute quantitation ( itraq ) , and mass defect ( isotope - differentiated binding energy shift tags , idbest ) . the nep2320 barocycler ( pressure biosciences ) was precooled with a circulating water bath to 4c prior to use . all extraction buffers were refrigerated ( 04c ) overnight prior to use and used cold . commercial protease and phosphatase inhibitor cocktails ( p8340 , p5726 , and p2850 , sigma aldrich , st . louis , mo ) were added to each buffer according to package directions immediately prior to use . pulse tubes were loaded according to the manufacturer 's instructions using 1.3 ml of the appropriate buffer in each tube . the tubes were immediately processed in the barocycler ( 20 cycles from 0 to 35,000 psi for 20 sec on each cycle ) . the resulting extracts were viscous and were treated with 25 l of micrococcal nuclease from staphylococcus aureus ( new england biolabs , ipswich , ma ) reconstituted at 1,000 units per ml per the manufacturer 's instructions , for 15 min on ice . the resulting extract was recovered with a transfer pipette and placed in a 2 ml microfuge tube along with any residual pellet . the pellets with hepes and td1 extraction buffers appeared as soft sticky disks generally pressed against the center frit of the pulse tube . significantly less pellet was formed in the td2 extraction buffer , and the td2 postextraction pellet proved to be very friable . insoluble materials were recovered from each extract by centrifugation ( 13,000 g for 15 minutes at 4c ) . the pellets were resuspended in 1 ml of td1 buffer by passing them through a 21-gauge syringe needle several times to disperse the pellet . both the pellets and clarified extracts were stored in aliquots at 80c prior to use . aliquots ( 45 l ) of both the hepes- and td1-clarified extracts were diluted into 365 l of proteosolve - ief buffer ( pressure biosciences , inc . ) . first dimension separation was carried out with 200 l of each diluted extract on the computer - controlled isoelectriq ief apparatus ( proteome systems , ltd . sydney , au ) using 11 cm , ph 310 readystrip ipg strips ( bio - rad laboratories , inc . separation was programmed with current limited to 50 a / strip in two steps : twelve hours on a concave voltage ramp set to start at 100 v and end at 10,000 v , followed by a constant voltage for 8 hours at 10,000 v. the strips were removed at 90 kv - h . the second dimension was performed on criterion 816% polyacrylamide tris - hcl precast gels in a dodeca cell ( both from bio - rad laboratories ) , equipped with the thermo - ec 57090 power supply at constant current of 60 ma / gel for 2 h. gels were fixed and stained with a proteomiq blue colloidal coomassie gel stain as described previously . gel images were acquired on a umax powerlook iii flatbed scanner as 48-bit color tiff files and converted to 16-bit grayscale using imagej software ( nih ) . image analysis was performed using ludesi redfin 3 software ( ludesi ab , malm , sweden ) . aliquots ( 17.5 l ) of each clarified extract and corresponding pellet suspension were denatured by the addition of 5 l of 4x nupage lds sample buffer ( invitrogen ) , 2 l of 1 m dithiothreitol , followed by heating to 90c for 10 minutes . the samples were centrifuged at 13,000 g ( 5 min ) and the entire contents run on precast 412% bis - tris nupage minigels , using the xcell surelock system ( invitrogen , carlsbad , ca ) , according to the manufacturer 's instructions . both seeblue and magimark ( invitrogen ) proteins were transferred to pvdf membranes at 65 v for 2 hours using a transfer buffer consisting of 20 mm tris , 160 mm glycine , and 0.04% sds . the pvdf membranes were blocked on a rotary shaker at room temperature with two different blocking buffers . a blocking buffer consisting of 100 mm phosphate buffered saline with 0.05% tween , 0.01% thimerosal , and 10% nonfat milk was used for the faslg , nrp1 , kdr , lamp-3 , bcl-2 , ccne2 , and akt blots . the blocking buffer used for the edg4 , edg2 , gpc3 , and tubb blots consisted of 25 mm tris , 0.15 m nacl , 0.1% tween-20 , and 0.01% thimerosal at ph 7.4 containing 2% nonfat milk . the second set of membranes were blocked for 1 h before addition of the primary antibody . in both cases , the blocking buffer used for incubation was removed before addition of fresh buffer with the primary antibody . primary antibodies were added at a nominal concentration of 1 g / ml in 10 ml of the respective blocking buffers for faslg , nrp1 , kdr , lamp-3 , bcl-2 , ccne2 , and akt blots , and a nominal concentration of 0.5 g / ml in 12 ml for the edg4 , edg2 , gpc3 , and tubb blots ( except with 1% nonfat milk ) . each blot was incubated with the primary antibody overnight on a rotary shaker at 04c . primary antibodies consisted of affinity - purified polyclonal antibodies against each biomarker purchased from various sources ( table 1 ) . appropriate cell lysates were used as positive controls for each of the antibodies in the western blots ( table 1 ) . after overnight incubation with the primary antibody , the blots were washed 4 - 5 times with their respective blocking buffers ( without the nonfat milk ) . washed blots were placed in 4 ml of the respective blocking buffer ( without the nonfat milk ) to which the appropriate antiprimary , secondary hrp - conjugated antibody ( cell signaling ) was added as supplied at a 1 : 1000 dilution for the faslg , nrp1 , kdr , lamp-3 , bcl-2 , ccne2 , and akt blots and 1 : 10,000 dilution for the edg4 , edg2 , gpc3 , and tubb blots . blots were incubated with the hrp - conjugated secondary antibodies for 1 h at room temperature on a rotary shaker . blots were again washed as described above and developed using supersignal west femto substrate ( pierce , thermo - fisher ) following the manufacturer 's recommendations . the chemiluminescent images were collected using a fluorchem sp gel imager ( alpha innotech , san leandro , ca ) . ovarian tumor samples extracted with td2 buffer using the barocycler ( as described above ) were diluted 1 : 10 in proteosolve - tdilute ( pressure biosciences ) containing both phosphatase and protease inhibitors ( previously described ) prior to any immunoaffinity work . the effect of the diluted td2 buffer on immunoassays was evaluated in several commercial elisa kits , including human transferrin kit ( bethyl laboratories , montgomery , tx ) , quantikine mmp-2 and mmp-3 ( r&d systems , minneapolis , mn ) , and pathscan total p53 and pathscan total akt1 ( cell signaling , danvers , ma ) . immunoassays were performed following the manufacturer 's instructions and in parallel with standards reconstituted in the diluted td2 buffer ( described above ) . rate assays ( change in absorbance with time ) were performed , instead of the standard single time point , to ascertain any residual effects of the td2 buffer components on the amplification step of the assay ( i.e. , modulation of horseradish peroxidase [ hrp ] enzyme kinetics or quenching of substrate color development ) . nonlinear least squares curve fit of the antibody binding ( 1 ) to the standards prepared in each buffer system was used to get the apparent affinity constants ( kaff ) . while the total antigen concentration [ an ] is known in the standard curve , the total antibody concentration [ ab ] and the affinity constant were determined simultaneously by nonlinear curve fit . the one sigma error of the estimate in each fitted parameter was determined from the jacobian matrix . ( 1)rate = k1+kaff[an]kaff[ab]2kaff+4kaff2[an][ab]+(kaff[an]kaff[ab]1)22kaff. the ovarian td2 extracts ( after 1 : 10 dilution in proteosolve - tdilute ) were also run in each assay to determine the effective biomarker concentrations in the extract . because of this dilution , the highest tissue concentration tested was 15 mg of tissue / ml , which in a 100 l elisa sample well is the equivalent of 1.5 mg of tissue . antibody - conjugated phytips ( phynexus , sunnyvale , ca ) were used for all immunoaffinity enrichment experiments . the experiments were conducted on a phynexus mea robot system ( phynexus , sunnyvale , ca ) using deep well plates . each tip contained 100 l fluidized beds of aminolink resin ( pierce protein research , thermo - fisher scientific , rockford , il ) conjugated to affinity - purified polyclonal antitransferrin antibody ( bethyl laboratories , montgomery , tx ) and packed in glycerol . the sample consisted of 1 ml of ovarian tumor extract prepared in td2 buffer and diluted ( 1 : 10 in proteosolve - tdilute ) as described above . nonspecific goat igg ( equitech - bio , kerrville , tx ) was added to the sample ( 9 mg per 10 ml of diluted sample ) to suppress histone binding to the antibodies present on the bead surface . glycerol ( used to pack and store the phytips ) was found to significantly alter flow through the phytips and suppress ionization in the subsequent mass spectrometric analyses . this glycerol was removed by washing the phytips with 100 mm pbs ( ph 7.2 ) in two stages using 96-well deep well trays ( seahorse labware , no . the first stage wash consisted of four successive quick rinses in single draw and expel cycle each of 1 ml at a 2 ml / min flow rate ( with 2 min holds at the top and bottom of each cycle ) . this removed the glycerol surrounding the bead bed , but left glycerol saturating the beads themselves . diffusion of the glycerol out of the pores of the beads in the second stage required four additional washes consisting of a 0.5 ml draw followed by 60 cycles of 0.3 ml volume at a 2 ml / min flow rate ( with 2 and 10 sec holds at the bottom and top of each cycle ) . this totaled about 30 min in each 20 ml wash volume ( for 12 tips ) . immediately following glycerol removal , the tips were immersed in a trough of an 8-row deep trough trays ( seahorse labware , no . s30020 , 32 ml per trough ) containing 10 ml of diluted tumor sample . next , 0.5 ml of sample was drawn into each tip , and 0.3 ml was cycled 240 times at 2 ml / min ( with 2 and 10 sec holds at the bottom and top ) . sample binding was immediately followed by a stringency wash in 4.17 m nacl with 83 mm napo4 ( ph 7.2 ) for 60 0.5 ml cycles at 2 ml / min ( with 2 and 10 sec holds at bottom and top ) . this was followed by buffer exchange into 150 mm pyridinium acetate ( ph 6 ) . five washes in 10 ml of pyridinium acetate in a trough ( 0.5 ml with 48 cycles of 0.3 ml each at 2 ml / min with 2 and 10 sec holds at the bottom and top of each cycle ) were required to diffuse all the salts from within the bead pores . antigens were subsequently eluted directly into 2 ml microfuge tubes containing 0.6 ml of 50% aqueous acetic acid ( 0.5 ml draw with 30 cycles of 0.3 ml at 2 ml / min ) . the residual pyridium acetate is a volatile buffer , which evaporated with the water in the speedvac . these were1.1 ml volume pipette tips packed by phynexus with 100 l of aminolink beads ( thermofisher ) in a fluidized bed configuration . affinity - purified , carrier - free , goat polyclonal antitransferrin antibodies ( no . a80 - 128a , bethyl laboratories , montgomery , tx ) were dissolved at 83 g / ml in 100 mm pbs ( ph 7.8 ) with 33 mm sodium cyanoborohydride ( nacnbh3 ) . a 0.6 ml quantity of the antibody solution was placed in the well of a deep - well plate for each tip . a 0.5 ml quantity of the antibody solution was drawn into each glycerol - free phytips ( washed as described above ) and processed for 960 cycles of 0.3 ml at 2 ml / min ( 6 h ) . unreacted aminolink aldehydes were then quenched with a 0.5 ml draw and 60 cycles of 0.3 ml each at 2 ml / min ( 0.5 h ) in 1 m tris(hydroxymethyl)-aminomethane chloride ( ph 7.8 ) with 33 mm nacnbh3 . residual tris and cyanoborohydride were removed with five washes ( 0.5 ml draw and 60 cycles of 0.3 ml each ) in 100 mm pbs , the last of which contained 0.05% sodium azide . an average of 35 g ab was bound to each phytip , as determined by uv280 absorbance change in the conjugation solution . the dried eluates ( described above ) were dissolved in 0.1 ml of 20 mm ammonium bicarbonate ( ph 8.2 ) , simultaneously reduced and capped by the addition of 2 l each of 2-vinylpyridine ( 50 mm in isopropanol ) and triethylphosphine ( 25 mm in isopropanol ) at 37c for 1 h , following the procedure described by hale et al . . after capping , the eluates were digested by adding 2.5 l of trypsin ( sequencing grade - modified , promega , madison , wi ) , reconstituted at 100 g / ml in 20 mm ammonium bicarbonate , for 2 h at 37c . addition of 0.11 ml of hplc grade acetonitrile quenched the digestion , and the digest was evaporated overnight in a speedvac . the pellet was resuspended in 25 l of maldi matrix ( -cyano-4-hydroxycinnamic acid dissolved at 5 mg / ml in 50 : 50 acetonitrile : water with 0.1% trifluoroacetic acid ) , and 1 l of the digest was spotted on a stainless steel maldi plate and analyzed using a q - tof premier ( waters , milford , ma ) . the resulting monoisotopic peptide peaks were selected using mmass , and matching proteins were identified using mascot to search the swiss - prot protein database . in order to show equivalence to classic extraction buffers , we performed a global proteomic analysis ( 2d gel electrophoresis ) using separate 100 mg aliquots of a cryogenically ground metastatic ovarian tumor sample pool ( sourced from multiple patients ) . using the barocycler , the first aliquot was extracted in hepes buffer and a second processed in the td2 buffer . the clarified extracts were diluted to 8.6 mg of tissue / ml in denaturing ief buffer for 2d gel analysis . comparison of the resulting gels by image analysis ( figure 1 ) reveals few differences in the more abundant protein species recovered . of 585 discrete protein spots identified , 97% were common in both position and abundance between the two gels . therefore , td2 buffer appears fully compatible with classic gel electrophoretic methods with little alteration in recovery of the more abundant proteins . only the most abundant proteins can be seen in coomassie - stained gels . thus , the above analysis tells us little about the quantitative extraction of membrane proteins . we , therefore , selected a number of representative biomarkers from different protein classes for more detailed analysis by western blots . in particular , we were interested in determining how much protein of each class was left behind unrecovered in the insoluble pellets . to this end , the insoluble barocycler pellets from each condition tested were recovered and treated by boiling in sds - page sample buffer . these sds extracts of the pellets were run side by side in western blots with the clarified extracts at similar effective tissue concentrations . tissue extractions were performed with hepes , td1 , and td2 buffers at 150 mg tissue / ml buffer concentrations . the barocycler extracts were centrifuged to recover an insoluble pellet and a soluble protein extract as separate samples . the extracts were diluted directly into 4x lds - page sample buffer ( invitrogen , carlsbad , ca ) to an equivalent gel loading concentration of 110 mg tissue / ml . all the pellets were resuspended in tdilute buffer to an equivalent concentration of 200 mg of tissue / ml to create a fine suspension . an aliquot of this suspension was diluted in lds - page sample buffer to an equivalent gel loading concentration of 140 mg tissue / ml . a series of western blots ( figure 2 ) were prepared from these extracts and pellets . were obtained from the same pulse tube ( i.e. , the same tissue preparation ) . therefore , it is possible to determine the relative abundance of each protein seen between the extract and pellet for each buffer . however , different pulse tubes are used for each of the different extraction buffers tested . because of water condensation during the weighing of frozen tissues , the amounts of tissue may vary between pulse tubes . however , recovery determinations between the clarified extract and its corresponding pellet are possible . detailed descriptions of the specific proteins analyzed by western blots ( figure 2 ) can be found in the supplementary materials available online at doi:10.1155/2012/838630 . key among these features are the number of transmembrane sequences , the theoretical ( sequence mw ) of the protein and any splice variants , and the reported measured mws of the protein , including any posttranslational modifications . two strong overlapping edg4 bands appear in the gel with nearly equal intensity and differing by less than 2 kda in weight in the cell line control ( lane j ) between 50 and 60 kda . single strong edg4 bands are seen in all the ovarian tumor samples at 55 kda , except the hepes extract ( lane b ) . three weak bands are seen in the hepes extract at 55 , 56 , and 57 kda , only one of which may correlate to edg4 . clearly , little edg4 is extracted into the hepes buffer in the barocycler since the amount extracted from the hepes pellet ( lane c ) is in great excess to any of the bands seen in the hepes extract . it is also possible that all the bands seen in this extract may be cross - reactive protein species because the band pattern is so different from that observed in any of the other samples . the strongest edg4 band in both the td1 and td2 extracts ( lanes d and f , resp . ) appears at a slightly lower apparent molecular weight than the edg4 band in the corresponding pellets . we note , however , that the molecular weights for the edg4 bands observed in the 1/3 dilution of the td2 extract ( lane h ) appear at the higher molecular weight observed for both the cell line control and the dominant edg4 band observed in the td1 and td2 pellets . therefore , we suspect that the edg4 protein in both the td1 and td2 extracts is running at a slightly lower molecular weight , either because the protein load in these lanes is too high or the sds fails to fully displace bound membrane lipids found in the insoluble pellet fractions . an additional weak band is seen at 56 - 57 kda in the 1/3 dilution of the td2 pellet ( lane h ) , but in none of the other samples , and may be an artifact . an edg2 band is seen in all samples at or just below 50 kda , including the cell line control ( lane h ) . the protein appears to run at a slightly higher molecular weight when recovered with hot sds from the barocycler pellets than when isolated from the barocycler extracts . this may reflect incomplete displacement of adsorbed lipids by sds from the insoluble protein found in the pellets . however , it may also merely reflect differences in protein concentrations between the gel lanes since the 1/3 concentration sample of the td2 barocycler extract ( lane h ) runs closer to the higher - molecular - weight band and appears less distorted . the edg2 band in the cell line control ( lane j ) is similarly distorted as the other extracts . edg2 recovery seems to improve dramatically from the hepes to td buffers . comparison of the td2 and td1 buffers in this sample shows only marginal recovery improvement . little or no faslg appears to be recovered in either the hepes or td1 extracts from the barocycler ( lanes b and d in figure 2 ) . faslg is seen in high abundance in the insoluble pellets from both of these extracts . however , nearly complete recovery of faslg is seen in the soluble td2 extract with little remaining in the insoluble pellet . other experiments ( data not shown ) suggested that faslg recovery was variable with td1 buffer , but was consistently high in the td2 buffer . no evidence of the 72 kda soluble form of nrp1 is seen on the western blots . the 140 kda membrane bound form is present in all extracts ( lanes b , d , and f ) . however , nrp1 recovery into the soluble fraction was lowest in hepes buffer with most of the protein found in the hepes pellet . about half of the nrp1 appeared to be recovered in the td1 extract in this experiment . however , nrp1 recovery in the td1 buffer was inconsistent between trials ( data not shown ) . almost complete recovery is seen in the td2 extract with only a trace of nrp1 left in the pellet . it is possible that the antibody used in these blots was reactive to a variant of the nrp1 protein that was not present in the cell line control used and which presents an epitope that is removed in the creation of the 72 kda soluble form . both the intermediate and mature kdr proteins are apparent in the western blot for both the cell line control and the tumor samples at 200 kda and 230 kda , respectively . partial recovery of the more abundant mature kdr protein is seen in hepes extract ( lane b ) with most of the protein left in the hepes pellet ( lane c ) . the intermediate glycosylated form was not seen in the hepes extract or pellet , possibly due to its lower solubility in the hepes buffer than either the td1 or td2 buffers . the aqueous solubility of the mature form is expected to be greater due to the higher level of glycosylation . most of the intermediate glycosylated form appears to be extracted into both the td1 and td2 buffers ( lanes d and f ) with apparently little left in either pellet ( lanes e and g ) . at least some of the more abundant mature form ( 230 kda ) was seen in all the extracts ( lanes b , d , and f , figure 2 ) and both the hepes and td1 pellets ( lanes c and e , figure 2 ) . the best relative kdr extraction was observed with the td2 buffer ( lanes f and g , figure 2 ) . two bands at 58 kda and 73 kda are seen for the lamp-3 protein in the western blot ( figure 2 ) . it is not clear if the upper band is a cross - reactive antigen or a hyperglycosylated version of the protein , but it is found in both the control cell line ( lane j , figure 2 ) and all tumor extracts . little of either molecular weight species are seen in any of the pellets , except for a trace of the 73 kda species in the td1 pellet . these high apparent recoveries , independent of extraction buffer used , may be due to the hyperglycosylation of this protein , particularly present between the transmembrane helixes . lamp-3 is found in all the extracts but appears to be in low total abundance overall in the tumor samples because long chemiluminescent substrate exposure times were required to visualize lamp-3 compared to the other biomarkers tested . reblotting with higher primary antibody titers did not appear to improve the signal strength ( data not shown ) . bcl2 is a single - pass apoptosis regulator predominantly found in the outer mitochondrial membrane , but also seen in the nuclear , and endoplasmic reticulum membranes . recombinant human bcl2 ( the first 218 amino acids and nonglycosylated ) is used as the antibody control . bcl2 recovery was good , but not complete , in all buffers tested , with the best apparent recovery in hepes buffer . however , because mitochondria are not expected to pellet at the 13,000 g used in this experiment , it is possible that bcl2 seen in the hepes extract is actually recovered from intact suspended mitochondria after heating in sds - page sample buffer . the same may be true of the other buffers , making it difficult to differentiate bcl2 recovery among the barocycler extraction buffers . in our work , two ccne2 bands are seen in the western blot of the cell line control and tumor samples ( figure 2 ) , a dominant band at 45 kda and a minor band at 47 kda . as ccne2 is not a membrane - bound protein , we would expect recoveries to be good in all the buffer systems tested . however , some of the dominant 45 kda protein remains in the hepes pellet ( lane c ) . a trace amount is also seen in the td1 pellet ( lane e , figure 2 ) . extraction appears to be nearly quantitative with the td2 buffer ( lanes f and g , figure 2 ) . these results could also suggest that one of the components of the td1 and td2 buffers may be assisting barocycler disruption of the nuclear membrane . when the western blot from our work is probed with a c - terminal - specific gpc3 antibody ( i.e. , raised against amino acids 303464 ) , a single strong band is seen in the gel at 30 kda for all the tumor and cell line samples . the gpc3 protein is lipid anchored to the cell membrane in this c - terminal region . none of these proteins is extracted into hepes buffer ( lane b , figure 2 ) . a small amount ( 10% ) appears to be extracted into td1 in the sample shown ( lanes d and e , figure 2 ) . about 75% appears to be extracted into td2 buffer ( lanes f and g , figure 2 ) . no other bands are seen in the blot probed with the c - terminal - specific antibody . when the blot is stripped and reprobed with an antibody specific to the n - terminal gpc3 region , however , a single band is seen near 80 kda in the cell line control ( lane j , figure 2 ) . the 30 kda fragment is not detected with the n - terminal - specific primary antibody . no corresponding bands in the 60 to 80 kda region are seen in the gel . since the mature protein is exported , it would normally be carried away from the tumor site in the blood ; therefore , little mature protein is expected to remain in the solid tumor . these results suggest that the 30 kda fragment may be the lipid - anchored c - terminus ( postmodification ) , which is only recovered in the td1 and td2 buffers and that the 80 kda band is the full - length protein with its lipid - anchored c - terminus intact . akt is seen at 60 kda in the western blot , as determined from the recombinant control ( lane i , figure 2 ) . the second band seen in the recombinant human akt control sample ( at 62 kda , lane i , figure 2 ) is attributed to incomplete cleavage of the his6 tag used in the purification of this fusion protein . akt recoveries are high in all the buffers tested , which would be expected for a soluble cytosolic protein , with no apparent differences between the buffers tested . while tubb is generally a cytosolic protein , it spontaneously forms dimers with alpha - tubulin ( tuba ) and is always in dynamic equilibrium between soluble a / b - dimers and polymerized microtubules , which can be insoluble depending on their size . a single strong band at 50 kda is seen in all lanes of the tubb western blot ( figure 2 ) . based on the relative chemiluminescent intensities , about 50% of the tubb is recovered in the hepes extract ( comparison of lanes b and c , figure 2 ) . td1 appears to extract more than 80% of the tubb present in the sample ( comparison of lanes d and e , figure 2 ) . td2 extracts better than 90% of all the tubb present . in separate time course experiments ( data not shown ) , we have shown that purified bovine tubulin ( cytoskeleton , denver , co ) remains soluble at 1 mg / ml in td1 buffer but polymerizes and precipitates nearly quantitatively within 24 hours in 20 mm hepes buffer . therefore , we believe that the lower apparent recovery of tubb in the hepes buffer is due to microtubule formation and precipitation in this extract . several initial attempts to process diced ( unground ) metastatic ovarian tumor tissue through the barocycler with the same extraction buffers did not produce good yields , even for cytosolic proteins ( data not shown ) . therefore , cryogenic grinding prior to barocycler extraction appears to be necessary . we speculate that the higher surface - to - volume ratio of the ground tissue allows for better access of the extraction buffer and shorter diffusional paths for the extracted proteins during pressure cycling . furthermore , as cited above , results with the td1 buffer proved inconsistent from sample to sample . some samples yielded small friable pellets and had good protein yields , and others appeared to leave a large sticky pellet after pressure cycling . the addition of a dispersion aid ( td2 buffer ) resulted in vastly improved sample - to - sample reproducibility . furthermore , no appreciable protein could be extracted from ground tumor samples heated at 95c for 1 h in either the td1 or td2 buffers in the absence of pressure cycling . when sds was added to these samples , in the form of sds - page sample buffer ( invitrogen ) , protein was subsequently recovered on additional heating ( data not shown ) . this demonstrates that pressure cycling is an integral part of the membrane protein extraction process with the proteosolve - td buffers and that the buffers alone do not act as detergents . several commercial sandwich elisa kits ( transferrin , mmp2 , mmp3 , akt1 , vegf , and svegf r2 ) were used to determine the effect of the td2 extraction buffer on subsequent immunoaffinity work . in each case , one vial of antigen standards was reconstituted as prescribed in the manufacturer 's instructions , and a second vial was reconstituted using the td2 buffer diluted 1 : 10 by volume in proteosolve - tdilute ( at ph 7.5 ) . otherwise , the kits were run as prescribed by the manufacturer with the exception that rate assays were performed to determine any residual effects of the td2 buffer on the reporter ( horseradish peroxidase ) activity or substrate color development . the small differences in affinity constants between the buffers used are within the experimental error of the serial dilutions and the curve fitting . furthermore , there was no consistent trend with kaff being either slightly higher or lower , depending on the assay , and generally within the expected preparation variation of the standards . the lone exception was the vegf assay , for which no assay response is seen for the recombinant human vegf165 standard reconstituted in the diluted td2 buffer . either this standard is not soluble or stable in the td2 buffer , or the buffer induces a change in the epitope recognized by the elisa antibodies . other than the anomalous vegf assay results , these data suggest that the td2 extraction buffer has no significant affect on antibody affinity or antigenicity of the recovered proteins . we expect , therefore , that better results may be obtained in each assay with further diluent optimization . the resultant antigen concentrations in the metastatic ovarian tumor td2 extracts were also determined in these assays and are summarized in table 3 . holotransferrin ( holo - tf ) is a surrogate marker for the blood or serum content of the tumor sample . serum tf concentrations are reported to be in the range of 2.94.0 10 ng / ml [ 58 , 59 ] . assuming all of the serum tf is recovered in the td2 extract , we estimate 0.5 10 ng tf per g of tissue , suggesting that the average blood / serum content of the frozen metastatic tumor samples is around 15% . matrix metalloproteinase 2 ( mmp2 ) is below the detection limits of the elisa ( i.e. , < 70 ng per g of tissue ) . matrix metalloproteinase 3 ( mmp3 ) is detectable above background at the highest extract concentration used , but is below the quantitation limits of the assay ( i.e. , 7 ng per g of tissue . no references to the tumor tissue concentrations of these proteins could be found , so recovery could not be determined . akt1 is a cytosolic protein for which the western blots ( figure 2 ) suggest nearly quantitative recovery with the td2 buffer . the total akt1 concentrations were determined to be about 20 g / g of tissue , determined from the single elisa sample tested . the glycosylated n - terminal domain , which is often cleaved , becomes a plasma - soluble biomarker species ( svegf r2 ) . assuming that the antibodies towards svegf r2 will crossreact with the membrane bound version , we thought this elisa kit might provide a more quantitative measurement of the amount of the membrane protein recovered than the western blot ( figure 2 ) , which suggested high recovery efficiency in the td2 extraction buffer . polanski and anderson report the plasma concentration of svegf r2 to be 15 ng / ml . the measured tissue concentration of vegf r2 is 1 0.1 ng / g of tissue , which is about twice as high as that expected for svegf r2 in the 15% blood contamination of the tissue sample . this translates to an expected svegf r2 concentration of 0.5 ng / g of tissue . however , the amount of svegf r2 measured for the hepes barocycler extract was indistinguishable from that seen in the td2 barocycler extract . yet , kdr ( vegf r2 ) recovery in hepes buffer was poor compared to that observed in the td2 extraction buffer ( figure 2 ) . possible explanations are that the elisa assay is truly specific to the soluble form of this protein , vegf r2 recovery differs negligibly between the different extraction buffers ( i.e. , the western blot results are not quantitative ) , or the amount of membrane - bound vegf r2 is low relative to that of the svegf r2 in the patients ' plasma . antitransferrin phytips were prepared as described in the methods . these were used to recover and enrich transferrin protein from the td2 ovarian tumor extract . elisa data indicate that the extract contains 80 g of transferrin ( table 3 ) . digest controls prepared with different concentrations of purified buffer - free apotransferrin ( sigma - aldrich ) suggested a limit of detection of about 4 ng of transferrin in a single maldi - ms spot ( 1 l of sample matrix ) for protein identification using peptide mapping . no improvement in sequence coverage was seen above 1 g of apotransferrin in a 1 l spot . of the 34 peaks found in the spectrum and submitted to a mascot search , 24 mapped to peptides from human transferrin , which was the top - ranked protein ( score of 280 ) . global proteomic comparison between the hepes and td2 extracts ( figure 1 ) shows few differences from among the higher - abundance proteins recovered from the sample . some 2,000 different proteins have titers greater than 5 10 copies in the average mammalian cell . the cellular titers of many cellular receptor ( membrane ) proteins are reported to be in the range of 1010 copies per cell , by comparison . the same dynamic range issues that plague global analysis of the plasma proteome also plague cellular proteomic analysis . with the limited dynamic range for a coomassie - stained gel , we may not see lower - abundance membrane proteins in such a global proteomic analysis . nonetheless , the 2d gel data are important in that they show virtually that all the same proteins are recovered in the td2 buffer as are recovered in a more standard aqueous buffer and in similar abundance . furthermore , the proteosolve - td buffers do not affect either the isoelectric focusing or sds - page separation coordinates of any proteins . therefore , we conclude that the proteosolve - td buffers are fully compatible with this time - honored global proteomic technique . the western blots ( figure 2 ) provide definitive evidence for the recovery of seven - different integral membrane proteins . edg2 and edg4 , both seven transmembrane g - protein - coupled receptors , appeared to be recovered well in the td2 buffer system and virtually not at all in the hepes control buffer . edg4 recovery , however , may not have been quantitative , but this is confounded by apparent cross - reactivity of the primary antibody with proteins of similar size . faslg , nrp1 , and kdr ( vegf r2 ) proteins , all single transmembrane proteins , were nearly quantitatively recovered in the td2 buffer but also showed partial recovery in the hepes control buffer . lamp-3 , an apparently low - abundance protein with four - transmembrane sequences , appeared to be recovered in good yield in all the extraction buffers . lamp-3 is highly glycosylated ( particularly between the transmembrane helixes ) , potentially improving its aqueous solubility . neither overnight incubation or boiling of the tissue samples in the proteosolve - td buffers showed any significant transmembrane protein recovery ( data not shown ) , suggesting that extraction of the membrane proteins was primarily due to the pressure cycling process . these observations support a pct mechanism ( a mechanistic discussion can be found in the supporting information ) in which the pressure cycle itself is primarily responsible for disruption of the lipid membranes and exclusion of the membrane proteins . the data further suggest that the proteosolve - td buffers need only to support the solubility of the pressure - extracted membrane proteins when the sample is returned to ambient conditions . what appears to be a 30 kda c - terminal domain , which contains the lipid - anchor , only appears to be recovered in the td1 and td2 buffers ( figure 2 ) . the soluble 65 kda mature gpc3 protein , resulting from cleavage of the c - terminal , lipid - anchored domain , was not seen in any of the samples . bcl2 , a mitochondrial protein with a single - transmembrane sequence , was the only protein to show better recovery in the hepes buffer than either td1 or td2 buffers . we believe that this was caused by the failure to pellet mitochondria during the centrifugation step ( 13,000 g ) . if mitochondria were left suspended in the extracts , then bcl2 would have been liberated from the membranes upon sample preparation for sds - page ( i.e. , boiling in sds sample buffer ) . higher recoveries of tubb were evident in both the td1 and td2 buffers compared to the hepes control . we believe this result is due to improved solubility of tubulin microtubules in the td buffers over that in hepes control . by contrast , the soluble protein controls ( i.e. , ccne2 , akt , and tubb ) appeared to be well recovered in all of the barocycler buffers . of particular clinical interest is that the td extraction buffers appear to stabilize membrane proteins in an aqueous environment that is compatible with subsequent immunoaffinity techniques ( e.g. , immunosorbent assays or immunoaffinity enrichment ) . with performance data from five different elisas ( figure 3 ) , we can say with good confidence that the td buffer system can have negligible effect on antibody affinity constants . nor does the td buffer system affect subsequent activity of the final elisa amplification reaction ( at least with the commonly used hrp enzyme ) . we believe this may be due to a structural difference in the vegf epitope in the proteosolve - td buffer system . this might be overcome by the selection of alternative capture or reporter antibodies for the elisa , but was untried . unlike the western blot data ( figure 2 ) , we found no significant difference between svegf r2/vegf r2 titers ( by elisa ) between the hepes and td2 extraction buffer systems ( table 2 ) . while membrane - bound vegf r2 ( kdr ) can be distinguished from svegf r2 in a western blot ( based on molecular weight differences ) , these can only be distinguished in an elisa based on the specificity of the antibodies , which are unknown in the kit used . this elisa was designed for svegf r2 ( a plasma marker ) and may not be cross - reactive with the membrane - bound version . however , the vegf r2/svegf r2 tissue titers determined ( 1 0.1 ng / g of tissue ) were double the svegf r2 titer expected to be present in the entrained blood in the sample . akt1 ( a soluble cytoplasmic protein ) was found to be present in the patients ' samples at a titer of 20 g / g of tissue . we note that both the mmp2 and mmp3 were below the limits of detection or quantitation of the elisas used in this study . it seems likely that many cellular proteins of clinical relevance may be similarly too dilute for direct measurement in the small ( 0.2 ml ) well volumes of standard elisa microwell plates . therefore , we investigated the use of immunoaffinity enrichment to concentrate lower - abundance biomarkers from larger sample volumes for subsequent analysis . subsequent mass spectrometric analysis was successfully used to confirm the identity of immunoaffinity - enriched transferrin ( figure 4 ) . not only does this enrichment experiment demonstrate the affinity and avidity of the tubb antibody in td2 buffer , but it also shows that none of the buffer components survive the enrichment process to interfere with enzymatic digestion of the sample , peptide ionization ( e.g. , ion suppression ) , or mass spectral analysis ( e.g. , adduct formation ) . immunoaffinity enrichment followed by direct mass spectrometric determination of the mass of the intact protein to identify possible clinically relevant isoforms was pioneered by nelson et al . and while we illustrate this method with a single protein ( tf ) to illustrate the method in this paper , however , we have applied it to enrich 33 different biomarkers from the same ovarian tumor samples ( data not shown ) . as mentioned previously , the td1 and td2 buffers are fully compatible with the common protein labeling chemistries ( data not provided ) . we have applied the described immunoaffinity / ms method using isotope - differentiated binding energy shift tags in our laboratory ( data not shown ) . this intact protein capture approach allows the detection of novel protein isoforms ( either sequence variants or posttranslational modifications ) that may be lost in other biomarker validation methods such as multireaction monitoring ( mrm ) or the use of stable isotope standards with antipeptide antibody enrichment ( siscapa ) . many solid tumors consist primarily of compact epithelial cells and connective tissue , which can be particularly recalcitrant to protein extraction . we note that the solid metastatic ovarian tumors used in this study had to be cryogenically ground to a fine powder before protein extraction proved effective . we also note that the results were variable with the td1 buffer , but with the addition of the dispersion they aid to create the td2 buffer , and this recovery variability was eliminated . we suspect that other softer tissues ( e.g. , liver ) or harvested cell lines may be processed via pressure cycling technology with either the td1 or td2 buffers and likely will not require prior cryogenic grinding . pressure cycling technology combined with the commercial protosolve - td extraction buffers appears to offer a new approach for protein , particularly membrane protein , extraction from tissues in a format suitable for subsequent clinical immunoaffinity methods and classic proteomic analyses .

integral membrane proteins play key biological roles in cell signaling , transport , and pathogen invasion . however , quantitative clinical assays for this critical class of proteins remain elusive and are generally limited to serum - soluble extracellular fragments . furthermore , classic proteomic approaches to membrane protein analysis typically involve proteolytic digestion of the soluble pieces , resulting in separation of intra- and extracellular segments and significant informational loss . in this paper , we describe the development of a new method for the quantitative extraction of intact integral membrane proteins ( including gpcrs ) from solid metastatic ovarian tumors using pressure cycling technology in combination with a new ( proteosolve - td ) buffer system . this new extraction buffer is compatible with immunoaffinity methods ( e.g. , elisa and immunoaffinity chromatography ) , as well as conventional proteomic techniques ( e.g. , 2d gels , western blots ) . we demonstrate near quantitative recovery of membrane proteins edg2 , edg4 , faslg , kdr , and lamp-3 by western blots . we have also adapted commercial elisas for serum - soluble membrane protein fragments ( e.g. , svegfr2 ) to measure the tissue titers of their transmembrane progenitors . finally , we demonstrate the compatibility of the new buffers with immunoaffinity enrichment / mass spectrometric characterization of tissue proteins .

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

PMC2684149

there are several advantages and disadvantages to diagnostic musculoskeletal ultrasound as compared to other imaging modalities . first , diagnostic us is estimated to be less expensive than mri . additionally , us is more patient friendly as claustrophobia , which may occur with mri scanners , is not encountered with us imaging . when compared to mri , patients with shoulder pain prefer diagnostic us . mri scans do have the advantage of examining a large area but may detect several abnormalities that may be clinically unrelated to the patient s complaints . diagnostic us also can examine large areas with extended field of view ( fov ) imaging , however the clinician can interact with the patient who can then direct the examination toward the symptomatic area ( fig . 1 ) . in this way , the clinician can focus the examination to the most relevant area . 1extended field of view ( fov ) imaging allows the clinician to capture large areas of tissue that may be clinically involved . here a longitudinal view of the entire medial gastrocnemius is viewed . ultrasound is an excellent modality for diagnosing injuries to muscle and tendon tissue extended field of view ( fov ) imaging allows the clinician to capture large areas of tissue that may be clinically involved . here ultrasound is an excellent modality for diagnosing injuries to muscle and tendon tissue us also has the advantage of being a dynamic study . for example , the affected part can be imaged in real time , observing for pathologic movement in tendon , bursa , muscles , or joints . unfortunately , mri does not offer this luxury , as there would be movement artifact distorting image quality . with diagnostic us , the patient simultaneously provides feedback and vital information to the examiner during the dynamic examination that may reveal tendon subluxation , joint subluxation , or ligamentous incompetence . since the diagnostic us exam is real time , the patient and even the referring physician can receive results immediately and then can outline a treatment strategy within the same visit . unlike mri , the traditional form of musculoskeletal imaging , , many specialties such as rheumatology , orthopedics , physiatry , and family practice are integrating this imaging modality into their daily practices . the portability of us machines makes this feasible . portable machines allow clinicians to examine patients in their offices , in the training room , and even on the field . outside of a mobile scanner , ultrasound offers advantages over fluoroscopy and ct scanning when utilized for interventional procedures . although fluoroscopy or ct scan can be helpful in localization of the structure to be targeted , both require ionizing radiation . fluoroscopy does not visualize soft tissues , relying on bony landmarks and often necessitates contrast in order to prevent inadvertent intravascular placement and to confirm placement . some patients may be allergic to contrast and therefore need prophylactic medication to prevent a reaction . therefore , various soft tissues and joints can be directly entered , aspirated , or drained . for example , when the piriformis muscle is injected with fluoroscopic techniques , it requires the use of pelvic bony landmarks to achieve correct placement , but without direct visualization it is not certain that the piriformis has been entered . however , recently smith has described a novel technique whereby the piriformis can be entered utilizing direct ultrasound visualization . this technique theoretically makes the rate of a false negative response much less . for more common injections it has been estimated that attempted intra - articular knee injections may miss the joint in up to 29% of the cases . with ultrasound guidance , the suprapatellar bursa , which communicates with the knee joint , can not only be examined for an effusion and synovial proliferation , but can be entered under direct visualization . blind subacromial bursal injections as well are known to have an inaccuracy rate of 2431% depending on the approach . with ultrasound guidance however , this bursa appears as a visible thin hypoechoic ( dark ) line overlying the rotator cuff tendons and can be easily injected from a lateral approach ( fig . 2 ) . in this way , ultrasound offers a more accurate and potentially more therapeutic interventional strategy to the musculoskeletal clinician than compared to blind subacromial injection . 2longitudinal view of the supraspinatus tendon with faint hypoechoic linear subacromial bursa ( arrow ) and overlying deltoid muscle longitudinal view of the supraspinatus tendon with faint hypoechoic linear subacromial bursa ( arrow ) and overlying deltoid muscle one limitation that diagnostic ultrasound has is its dependence on body habitus . for instance , a 12 mhz linear array transducer can visualize very superficial structures with high resolution , but imaging of a hip joint or rotator cuff in an obese or extremely muscular individual can be extremely limited . although recent advances have improved high frequency linear array transducers , a lower frequency curvilinear transducer ( 35 mhz ) may be needed to provide adequate penetration for deeper structures . with greater depth of penetrance , though , resolution can be sacrificed , making musculoskeletal us limited as a modality in obese or muscular patients . however , recent advances in tissue harmonics have improved visualization and resolution of deeper structures even in these challenging cases [ 3 , 9 ] . ultrasound involves the reliance of placing the transducer and hence the beam at a 90 angle to the structure being imaged . any deviance from this will result in the reflection of the beam away from the transducer , causing a reduction in the echogenicity ( brightness ) of the tissue being examined . this artifact is referred to as anisotropy and can be eliminated by maintaining the beam perpendicular to the involved tissue ( fig . good technique also involves maintaining adequate skin contact , confirming the presence of pathology in orthogonal planes , and using the appropriate transducer size for the specific situation . 3(a ) transverse view of the proximal long head of the biceps in the bicipital groove . now the transducer is not 90 degrees to the tendon , causing it to appear hypoechoic ( darker ) . this may mimic a tear of the tendon and is referred to as anisotropy ( a ) transverse view of the proximal long head of the biceps in the bicipital groove . now the transducer is not 90 degrees to the tendon , causing it to appear hypoechoic ( darker ) . the evaluation of tendon pathology is probably the most common clinical indication to obtain a diagnostic musculoskeletal ultrasound . on us , normal tendon , which is composed of fascicles of collagen fibers running in parallel , appear as fibrillar hyperechoic ( brighter ) bands . in normal conditions , there will also be a flat hypoechoic structure surrounding the tendon , representing a synovial sheath containing a small amount of fluid . an exception to this is the achilles tendon , which has a closely adherent paratenon and is often normally imperceptible . in cases of tenosynovitis exceptions do exist . a significant amount of fluid surrounding the bicep may indicate primary bicipital tendonitis . however , fluid around the biceps tendon may be a secondary sign of a complete rotator cuff tear as fluid communicates with the glenohumeral joint through the subacromial bursa . many articles have been written defining criteria for partial and full thickness tears , the sensitivity , and specificity of diagnostic us , as well as the potential pitfalls associated with this modality [ 1214 ] . reports vary with the sensitivity as high as 100% for full thickness tears , but similar to mri , us has a much lower sensitivity for partial thickness tears . several signs that indicate a full thickness rotator cuff tear include nonvisualization of the cuff , discontinuity of the cuff , cartilage interface sign , and interposition of the subacromial bursa ( fig . 4 ) or deltoid into the vacant tendon [ 12 , 13 , 15 ] . criteria for determining partial cuff tears or tendonosis , like that of mri , are somewhat controversial . these signs may include a thickened relatively heterogenous appearing tendon , cortical irregularity , as well as a defect in the cuff tendon that does not communicate fully through from the bursa to the articular side [ 1517 ] . this is actually the subacromial bursa which has filled in the defect created by the cuff tear . ( photo courtesy of jay smith , md ) full thickness supraspinatus rotator cuff tear ( arrow ) . this is actually the subacromial bursa which has filled in the defect created by the cuff tear . ( photo courtesy of jay smith , md ) the achilles tendon is another excellent structure well - defined with diagnostic us . the normal tendon thickness seen in cross - section ( axial or transverse view ) is approximately 56 mm [ 18 , 19 ] ( fig . 5 ) . most pathology , including tears and tendonopathy occurs approximately 26 cm from the achilles insertion . however , a potential pitfall exists when the nearby medial plantaris tendon can be mistaken as partially intact achilles tendon when in fact a complete tear is present . 5transverse view of the achilles tendon at the level of the posterior malleolus transverse view of the achilles tendon at the level of the posterior malleolus danielson et al . power or color doppler enables the examiner to identify these abnormal penetrating vessels in cases of achilles tendonopathy [ 23 , 24 ] . if this strengthening program does not resolve the patients complaints , alfredson has reported that injecting a sclerosing chemical into these aberrant vessels under us guidance results in normalization of the achilles tendon and significant reduction of clinical symptoms . small tendon tears on initial examination may go undetected . with sonopalpation or motion , further tendon separation may become apparent . owing to hematoma formation and associated debris , a complete achilles tendon tear may be poorly demarcated . yet with ankle dorsiflexion , a discontinuity will be more easily demonstrated . real time dynamic subluxation of tendons can not be visualized with current mri technology . with us however , biceps , peroneal , or posterior tibial subluxation or dislocation can be visualized with dynamic maneuvers . for the biceps , this may involve elbow flexion combined with forearm supination and glenohumeral external rotation . in the ankle , peroneal tendon subluxation over the lateral malleolus can be demonstrated with combined active ankle dorsiflexion and eversion . a common elbow injury seen in overhead athletes , namely baseball pitchers , is a tear of the ulnar collateral ligament ( ucl ) . this ligament ( fig . 6 ) normally resists the tremendous valgus forces that occur at the elbow during an overhead throw . overtime , especially in pitchers , the anterior band of the ucl becomes lax and may rupture due to the tremendous valgus forces applied to the elbow during the throwing motion [ 28 , 29 ] . musculoskeletal ultrasound is an excellent imaging modality to clearly define the extent of an injury . applying a valgus force during the examination , which simulates the force during the throw , has been shown to be an added benefit of this imaging modality in diagnosing ucl laxity . fig 6a normal appearing ulnar collateral ligament of the elbow ( arrow ) a normal appearing ulnar collateral ligament of the elbow ( arrow ) another common athletic ligamentous injury involving the ankle joint is a tear of the anterior talofibular ligament and if more severe , the calcaneal fibular ligament . although ultrasound can not detect underlying bone edema , it can aid in grading the severity of the tear , which may be helpful for prognosis and return to play . like the elbow , applying additional stress may also aid in determining severity of ankle ligamentous injury . although it can not detect intra - articular knee pathology such as meniscal or cruciate tears adequately , ultrasound can easily visualize the medial , lateral , and patellar ligaments quite readily . particularly when combined with dynamic stress testing , us has been proven to be a sensitive test for detecting medial collateral ligament ( mcl ) tears . the normal mcl is composed of a hyperechoic superficial and deep band separated by a hypoechoic layer representing loose areolar tissue . in partial mcl injury or sprain , thickening of the ligament will occur and the superficial and more commonly , the deep band will appear with decreased echogenicity . complete rupture of the ligament will appear as an interruption of the hyperechoic bands within the ligament and there can be an associated fluid collection . though plain radiographs and mri are useful at assessing intra - articular and periarticular pathology , us can add a complementary role for these imaging modalities . for example , us is undeniably the best imaging modality for detecting small joint effusions , which are indicative of underlying joint pathology . in fact , effusions as small as one ml can be identified with diagnostic us ( fig . 7 ) . with these small effusions it may be difficult to blindly aspirate the joint . in these cases , us helps not only to locate the presence of an effusion but also serves as a guide for aspiration . 7normal appearing longitudinal view of the hypoechoic ( dark ) suprapatellar bursa ( arrow ) underlying the quadriceps tendon . the patella is the hyperechoic white structure to the left and the femur is the hyperechoic structure towards the bottom of the image . an effusion would be visible in this location normal appearing longitudinal view of the hypoechoic ( dark ) suprapatellar bursa ( arrow ) underlying the quadriceps tendon . the patella is the hyperechoic white structure to the left and the femur is the hyperechoic structure towards the bottom of the image . an effusion would be visible in this location us can also aid in detecting the cause of the underlying effusion . although mri is superior at visualizing intra - articular pathology , color or power doppler can detect concomitant increased blood flow detected in the synovium of inflammatory or infectious arthritis . the synovium in infectious or inflammatory arthritis is thickened , hypertrophic , and edematous , appearing as a hypoechoic band between muscle or fat [ 35 , 36 ] . most infectious effusions also have some component of echogenicity but may also give the appearance of a compressible hypoechoic mass . evaluation of large synovial joints is most easily performed at the suprapatellar recess of the knee , anterior synovial recess of the hip , and posterior synovial recess of the shoulder . measuring the thickness of the synovium in inflammatory arthritis has been shown to be a reliable means of following the effectiveness of therapy [ 37 , 38 ] . articular hyaline cartilage appears as a thin , hypoechoic line juxtaposed to the subchondral cortical bone . early ultrasonographic findings compatible with cartilage pathology , in particular inflammatory and osteoarthritis , include edema . with edema there will be an increased thickness of the articular cartilage with inhomogenity and an ill - defined cartilage margin . comparison with the opposite side may be helpful to obtain a baseline , however arthritic conditions are often symmetric . chondral and osteochondral defects , which can occur through trauma , infarction , or osteonecrosis , can also be detected as loose bodies . fibrocartilage , such as that found in the knee menisci , is composed of densely packed collagen fibers with interposed chondrocytes . this infrastructure is responsible for the homogenous hyperechoic appearance seen on us . though us can not penetrate into the joint proper to directly visualize cartilaginous injuries , there are secondary signs that may indicate underlying cartilage injury . meniscal cysts in particular are most commonly located on the lateral joint line and often communicate with horizontal meniscal tears . they are frequently seen as hypoechoic , and occasional anechoic ( without echogenicity ) structures adjacent to the meniscus and often require surgical intervention for treatment . labral tears of the hip or shoulder can be identified using diagnostic musculoskeletal us , particularly if the defect extends to the peripheral joint margin where the cartilage can be examined . though mr arthrography remains the gold standard , it is an invasive and expensive procedure . with us , a non - invasive and relatively inexpensive test , there are a number of findings that indicate the presence of underlying labral pathology . paralabral cysts of the hip or suprascapular ganglia are associated with concomitant labral pathology of the hip and knee joints , respectively . van holsbeeck has reported that us is particularly useful if used immediately after a dislocation . the intra - articular hemorrhage serves as a natural contrast medium and improves direct imaging of a labral tear . however , until further research is performed utilizing us , mri arthrography would still be considered the gold standard for imaging labral injuries . the portability , ease of use , and superior spatial resolution make ultrasonography an excellent imaging modality for detecting and classifying these injuries . additionally , ultrasound can also identify non - traumatic or primary muscle pathology such as myositis [ 24 , 40 ] . occasionally patients are unable to completely localize the area involved especially when it involves a large muscle group such as the biceps femoris . , the clinician has to follow obliquely running muscles like the sartorius on various cuts and sequences . however , the musculoskeletal ultrasonographer can follow this muscle from its origin to its insertion during one scan . also if a muscle and its tendon is torn and retracted , mri may not identify the location of the entire tendon . for example , in a complete quadriceps tear , standard knee mri protocol may not include the distal torn portion . however , us offers the ability to track the torn portion proximally and is helpful in measuring the degree of retraction . muscle tears can result from either direct or indirect trauma . in direct muscle injury , often there is significant contact , compression , and resultant destruction of muscle fibers . on us , these injuries are characterized by an irregular cavity with shaggy borders . often this cavity may contain a hematoma that may limit complete evaluation but after 23 days the hematoma becomes anechoic , allowing true estimation of the injury . a complication of this direct trauma can be myositis ossificans ( fig . 8) . with indirect muscle trauma there is often an eccentric injury , which results in a tearing of muscle fibers at the myotendinous junction . tears require good imaging technique with orthogonal views in the longitudinal and axial planes to properly identify the defect . there may also be a hypoechoic gap identified with gentle transducer pressure is compressible , reproducing the patient s pain . this technique is often referred to as sonopalpation and is another advantage associated with us . the degree of residual fibrous scarring may also help to predict the risk of recurrent injury . 8longitudinal view of myositis ossificans ( arrow ) located deep within the vastus intermedius of a 40-year - old male basketball player . four weeks prior to this , another basketball player s knee struck this athlete in the thigh that resulted in persistent pain with end range knee flexion longitudinal view of myositis ossificans ( arrow ) located deep within the vastus intermedius of a 40-year - old male basketball player . four weeks prior to this , another basketball player s knee struck this athlete in the thigh that resulted in persistent pain with end range knee flexion one of the most common studied peripheral nerve entrapments is carpal tunnel syndrome ( cts ) . in this condition , typically the proximal portion of the nerve becomes swollen while the portion coursing through the tunnel is compressed . in one study , cross - sectional area of greater than 10.5 mm was compatible with electrophysiological abnormalities seen on nerve conduction studies . additional abnormal us findings seen in cts include a decrease in median nerve echogenicity and loss of the normal fascicular pattern . with more severe cases , there may also be an increase in blood flow within the nerve on color doppler . though us can be useful to help guide a therapeutic steroid injection near a neural structure for pain relief , a randomized study comparing it to blind injections has not been performed . however , by directly visualizing peripheral nerves , inadvertent injury can be avoided during injections . morton s neuroma is an abnormal fibrous condition of the digital nerve most commonly located between the 3rd and 4th and second and third web spaces of the feet and may produce pain and paresthesias of the respective toes . when a neuroma is present , us examination of the plantar surface between the metatarsal heads will reveal an ill - defined , poorly reflective ovoid or fusiform mass measuring 57 mm in diameter [ 19 , 45 ] . us can be additionally helpful in this painful condition since it can help guide a local steroid injection for pain relief . bursae are sac - like structures that facilitate movement of musculotendonous structures and are optimally visualized with diagnostic us . inflammation of these structures , commonly due to increased friction or trauma , can become a source of pain and dysfunction . the more common clinical conditions associated with these structures include subacromial , greater trochanteric , pes anserine , and olecranon bursitis . normally , these structures on us appear as a thin hypoechoic line no more than 12 mm in height with hyperechoic boundaries reflective of a fluid tissue interface ( fig . 2 ) . when enlarged , these bursae may be mistaken for soft tissue tumors yet they are fluid filled and therefore often compressible . , the synovial walls of the bursa may become thickened with proliferative tissue and may have associated calcifications and internal hyperechoic debris . the differentiation between infectious , metabolic , or inflammatory bursitis in these cases may be difficult , but us guided aspiration for fluid analysis helps decipher this clinical conundrum . recent improvements in technology allow one to image various structures including tendon , muscle , joints , and even nerve with excellent resolution . portability allows examination not only in the office but also in the training room and playing field . low cost , real time imaging , and its ability to be used as a guide for interventional procedures make this imaging modality ideal for most musculoskeletal clinicians .

musculoskeletal ultrasound ( us ) can serve as an excellent imaging modality for the musculoskeletal clinician . although mri is more commonly ordered in the united states for musculoskeletal problems , both of these imaging modalities have advantages and disadvantages and can be viewed as complementary rather than adversarial . for diagnostic us , relative recent advances in technology have improved ultrasound s ability to diagnose a myriad of musculoskeletal problems with enhanced resolution . the structures most commonly imaged with diagnostic musculoskeletal us , include tendon , muscle , nerve , joint , and some osseous pathology . this brief review article will discuss the role of us in imaging various common musculoskeletal disorders and will highlight , where appropriate , how recent technological advances have improved this imaging modality in musculoskeletal medicine . additionally , clinicians practicing musculoskeletal medicine should be aware of the ability as well as limitations of this unique imaging modality and become familiar with conditions where us may be more advantageous than mri .

Advantages and disadvantages of diagnostic US Tendon pathology Ligament Joints Muscle Nerve Bursae Summary

PMC4412514

the siege of krishnapur takes place in a fictional east india company station as it is suddenly caught up in the events of the mutiny in the summer of 1857 . the plot concerns the efforts of the british residents , particularly the collector , mr . hopkins , an adherent of progress and industry obsessed with the great exhibition of 1851 , and the byronic poet george fleury , to defend the garrison long enough for relief to arrive . despite the relative simplicity of the novel s plot , to consider farrell an adherent of the realist imperial adventure novel in the style of his near contemporary john masters would be to overlook the ironic and parodic intent of his work . farrell s objective in the siege of krishnapur , as he revealed in an interview shortly after the novel s publication , was to create a novel of ideas which could be read at the same time simply as an adventure story ( dean 1973 , 11 ) . unlike a novelist such as masters , farrell s work does not seek to present the values of empire as a curative for the social ills of the 1970s . instead , the siege of krishnapur is a novel about the continual clash of opposites : one of past and present , tradition and progress , the british empire and india , and the englishmen and indians . in order to explore both why and how farrell would compose his novel in this way , it is important to preface analysis of the siege of krishnapur with some contextual information on the literary and cultural circumstances in which it was written . though the significant historical and historiographical approaches of the post - war period are often linked to the so - called cultural turn of the late 1960s , it was in the 1970s that the process of re - examining the modern and imperial past of britain began in earnest . however , rather than this process of re - examination diminishing literary interest in history and the historical novel , the booker prize records are testament to the reverse ; indeed , as a.s byatt asserts in on histories and stories : selected essays , there was a sudden flowering of the historical novel in britain in the post - war period , representative of a wider authorial and public fascination with the rapidly fading world of britain s imperial grandeur ( 2000 , 9 ) . mariadele boccardi argues that this trend is a reaction against post - structuralist and postmodern arguments for the end of history ; however , given the destructive character that pervades farrell s work and recurrent use of illness as metaphor , his engagement with britain s imperial past is wholly anti - nostalgic and one intent on re - examining history with as much scrutiny as either of these movements ( 2009 , ii ) . similarly , farrell s decision to write a mutiny novel twenty - six years after indian independence means that it would be unwise , not to mention difficult , to separate entirely his work from the influence of contemporary scholarship of the 1970s . it has been argued by critics such as peter morey that farrell is a postmodern author returning to british india to criticise colonialism through the blending of comedy and pathos ( 2000 , 110 ) . given the politicised nature of farrell s undertaking as well as the inherent and inescapable racial context of empire and british india , a range of postcolonial theories and analyses can be applied to the siege of krishnapur as well , in particular those that explore racial stratification and the classificatory techniques of british colonial society . however , such analyses must also be approached with caution so as not to erroneously and anachronistically label farrell s work wholly within postcolonial discourse . for whilst the siege of krishnapur is sensitive to the portrayal of the colonised subject , the novel draws its focus largely on the actions , thoughts and failings of the coloniser , that of the british garrison ; again with emphasis on the temporal duality of farrell s novel , this focus is intended as a synchronic critique of empire and the popular conception of a key moment in imperial mythology , namely the effect of colonization on the colonising power itself in both short and long term ( crane and livett 1997 , 99 ) . thus farrell s novel sits uneasily between paradigms ; too parodic to be sincerely postcolonial and too sincere to be entirely postmodern . it should be recognised though that farrell s return , after empire , to the days of colonial india in no way validates colonialism s existence but rather exposes its limitations . this intent means that farrell s post - colonial return is not merely temporally and aesthetically after - empire but engages in a critical pursuit of its legacy , quite literally going after empire through parodic and ironic critique . in addition to postmodernism and postcolonialism , the other key theme of many historical novels of the 1970s was that of unrest -- a direct response to the political turmoil of the decade . in his seminal the break - up of britain crisis and neo - nationalism , 196575 ( 1979 ) , tom nairn asserted that rapid decolonisation , economic stagflation and the social and political conflict that characterised the 1970s were representative of a society undergoing a slow - motion landslide of disintegration ( 62 ) . bart moore - gilbert s analysis of 1970s fiction in which he argues that british fiction of the era gives the impression of a society on the verge of social disintegration , or civil war seems particularly applicable to farrell whose novels of the period all feature episodes of violent unrest and upheaval ( 1994 , 152 ) . farrell s empire trilogy repeatedly features episodes in which the british societies depicted are on the verge of moral and physical disintegration . examining the root causes of british decline is the driving force of farrell s narrative in the siege of krishnapur , and the novel illustrates a colonial culture preoccupied with notions of its own progress , primarily in medical , scientific and spiritual matters . farrell s choice of period , location and medium in which to conduct this examination is highly significant , as it represents the high - water mark of the british imperial mission to india and its simultaneously civilising and industrialising intent ( harrison 2002 , 154 ) . farrell s critical approach thus becomes an attack on british naivet as well as a cherished myth of empire and nationhood . the irony of the british clinging to their noble principles and certainties of pre - eminence whilst under siege is exploited to great effect by farrell throughout ; indeed , some of the book s most blackly comedic moments come as its characters extol empire s virtuous intent as their world quite literally falls to pieces around them amidst the sepoys cannonades . the language used to describe the perceived spread of victorian enlightenment is commonly that of a medicalised , often physiological nature ; as fleury , who has travelled to india to research a tract on how empire has hastened the development of native civilisation , states : besides doctor , everyone i talk to in calcutta about my book tells me to look at this or that a canal that has been dug or a cruel practice like infanticide of suttee which has been stopped and these are certainly improvements of course , but they are only symptoms , as it were of what should be a great beneficial disease ( farrell 2008 , 38 ) . farrell s use of medical idiom here is not just a literary motif but rather establishes medicine as a key component of the british presence in india , its authority bound up in scientific knowledge and its responsibility to improve the lives of its indian subjects . for a novel so clearly critical of empire , farrell s own narrative voice is far more muted than one might imagine when it comes to the subject of british folly . rather than choosing to ridicule openly his characters for their misguided beliefs with a direct authorial voice , farrell allows the peculiarities of historical fiction to accomplish this for him . in order to give his satire its teeth , farrell chooses to employ a range of eyewitness accounts and factual medical material as part of his unpicking of british history . for instance , krishnapur , though an invention , is a portrait drawn from farrell s visits to lucknow and cawnpore in 1971 as detailed in his india diary , which was posthumously published along with his unfinished novel the hill station in 1981 . afterword in the siege of krishnapur in which he reveals his extensive use of diaries and letters from the siege of lucknow ( june - september 1857 ) , often repeated nearly verbatim in the text of the novel or whose authors serve as inspiration for many of his characters ( mcleod 2007 , 61 ) . chief among the accounts farrell employs are those of the reverend henry s. polehampton and maria germon , wife of an army officer , both present during the siege of lucknow , and the testimony of mark thornhill , company collector at muttra ( farrell 2007 , 314 ) . these figures , their testimonies and their experiences of privation , sickness and endurance recur throughout farrell s novel , not only providing the plot with dramatic set - pieces but also supplying a great deal of the grim comedy which pervades the narrative . much like his use of the narrative accounts of the indian mutiny , farrell uses contemporary sources such as medical diaries , scholarly journals , scientific case - studies and newspaper articles from the 1850s as the basis for his text , reflecting their tone , style and structure throughout . malcolm dean relates that he found nineteenth - century copies of the british medical journal among farrell s papers , indicating he took pains to make his portrayal of disease as authentic and period - specific as possible ( farrell 1993 , 197 ) . such research emphasises the relevance of farrell s novels to a changing medical climate . farrell s literary critique of empire corresponds with the contextual emergence of the medical humanities ; ronald a. carson has argued that the medical humanities are a product of the turbulent 60s , when authority and expertise were being questioned and traditional ways of doing things were being challenged ( 2007 , 322 ) . farrell , writing in the 1970s , conforms to this view with the siege of krishnapur in particular seeking to question the traditional narratives of the british presence in india and the perception of authority in british medical expertise . further , in his unpublished diary farrell records that he became interested in writing , largely i suppose as a sort of self - therapy ( farrell 1965 , 69 ) . farrell s novels are therefore not only relevant within their own context , both as a means of understanding how he came to terms with his own illness through literature and how the british nation sought to come to terms with the rapid decline of empire by the same means , but are also relevant today as examples of how that process involved the intersection of medicine and literature on a textual and metatextual scale . in the imprint of recorded events in the narrative form of j. g. farrell s the siege of krishnapur ( 1993 ) , lars hartveit argues how the appeal of documents naturally intersected with farrell s literary intent and chosen genre . indeed , in his own archival research , conducted after malcolm dean s , hartveit noted a file card marked health among many others within farrell s papers at trinity college dublin , indicating his inclusion of medical material was in no way an incidental detail of his writing but rather a systematic and deliberate process of inclusion ( 1993 , 458 ) . farrell s reasoning behind such documentary faithfulness was his belief that history leaves so much out it leaves out the most important detail of all : what being alive is like ( 1993 , 452 ) hartveit s analysis is based on the work of metahistorian hayden white and , as such , he views farrell s decision to construct fiction from historical detail as a matter of ordering material in a different way , thereby equating the novelist and historian as two - sides of a narrative coin ( 1993 , 452 ) , further suggesting farrell s status as a multidisciplinary writer . however , farrell s editorial decisions as to what to include in his narrative are often in excess of the then traditional historian of empire , and it is only on further inspection that the full extent of farrell s so many of farrell s textual details and vignettes are repeated verbatim from his sources ; from polehampton , farrell takes a liver - coloured spaniel , a fallen woman in a dak bungalow and an argument between a catholic and church of england over a group of unidentifiable bodies and their burial rites ( polehampton 1858 , 1089 ; 113115 ; 311 ) . from thornhill , he repeats the confusion over the sudden appearance of chapattis in the spring of 1857 ( thornhill 1884 , 2 ) . from germon , he derives boils , apoplexy and the bidding of exorbitant sums on everyday items as food runs scarce ( germon 1870 , 55 ; 8384 ) . folding these details into the siege of krishnapur , as he does with similar primary sources in his other novels , illustrates farrell s feeling that conventional , documentary history is somehow inadequate or deficient in that its ' factual ' demands means it lacks the emotional and physical elements literature is able to provide . rather , the novel is more readily disposed to represent the effects and subjective experience of living through history or , as farrell put it , undergoing history, becoming more accessible as a result ( mcleod 2007 , 37 ) . recognising as subsequent critics have that the british experience of india was intensely physical ( collingham 2001 , 1 ) , the chief means through which farrell achieves this is through the embodiment of experience : the positioning of the body , its deficiencies and its treatment at the centre of his novels . the effect of this process of inclusion is that through individual incidents , objects or identities , farrell is able to criticise more than the artform or profession in the foreground ; the technique enables him to critique the wider ideology and values of empire in simultaneity . in crane and livett s troubled pleasures , this process is labelled interdiscursivity ( 1997 , 87 ) . crane and livett relate interdiscursivity to the novel s penultimate chapter in which the relief force arrives at krishnapur and the survivors are led away from the compound , comparing farrell s portrait with the real - life painting the relief of lucknow ( 1859 ) by thomas jones barker . however , the principles of interdiscursivity pervade many other scenes and chapters throughout the book , not especially those which feature medicine and the role of the doctor . as the following sections will illustrate , farrell uses medical details as part of this process of interdiscursivity , moving his novels away from orthodox historical fiction and towards medical humanities ; by employing medical treatment and practice in an imperial adventure novel , farrell not only criticises the historical record of empire but attacks the foundation of science , medicine and reason on which the garrison of krishnapur and empire are based . among the expected instances of scurvy , malnutrition , amputation and nervous exhaustion that befall the garrison of krishnapur , the two central subjects that form the bulk of farrell s medical discourse are that of cholera and phrenology . the inclusion of infectious diseases and pseudoscience within the novel are not , however , merely incidental plot points or means of inserting instant authenticating detail ; instead the inclusion of nineteenth - century debates over cholera transmission is integral to farrell s novel , a fact made clear in his personal papers and manuscripts housed in trinity college , dublin . in farrell s handwritten notes and drafts for the siege of krishnapur , he provides an early list of key scenes and plot points around which the novel s narrative will pivot . included alongside headings such as the last great battle scene and the relief is the heading the great cholera controversy scene, an indication of farrell s own assessment of how important the scene was within the novel s structure and critical intent . tensions and disagreements between characters regarding cholera simmer throughout the narrative as the contagion steadily spreads , intersecting with deliberately anachronistic debates over darwinism , the potential for a moving daguerreotype and other innovations . however , unlike these sections of dialogue , which add humour and contribute to the novel s warping of history and chronology , the great cholera debate is strictly factual and period - accurate . alongside the index card health noted by hartveit , farrell s papers contain another entitled cholera and a range of photocopied , transcribed and typed material on medicine and health drawn from a variety of nineteenth - century sources . farrell s reading lists and notes are extensive and varied ; indeed , both dean and hartveit make reference to farrell s research ethic when preparing a new manuscript , particularly how his research in the british library and the victoria and albert museum archive would take at least a year ( hartveit 1993 , 451 ; farrell 1993 , 195 ) . firstly , the decision to ensure that his fictional portrayal of the indian mutiny was rooted in a factual basis indicates a desire for exactitude and authenticity on farrell s part that went beyond the traditions of his chosen form , the imperial adventure story . this decision also complicates farrell s categorisation under either postcolonial or postmodern genres , as his use of this material in his novels neither fits wholly within linda hutcheon s definition of historiographic metafiction ( 1988 , 14 ) , nor does it explicitly privilege subaltern voices ( spivak 1989 ) . secondly , the conflation of fictional and factual modes of writing , especially in his use of case notes from medical procedures and the language and idiom of nineteenth - century medical practice , means that farrell s novel represents an interface between medicine and literature and situates him within the discourse of medical humanities . the focus on cholera within the siege of krishnapur is a means through which farrell is able to consider resistance to change in both a contemporary and historical setting specifically through a medical lens . farrell uses the conflict over cholera to illustrate a range of internal divisions and disagreements amongst the novel s british characters . as the siege continues , the general rivalry between the garrison s civil surgeon dr . mcnab becomes more greatly pronounced , coming to a head over how best to treat cholera . when the collector takes a tour of the sick bay , dunstaple becomes enraged over mcnab s methods and demonstrates what he believes to be the proper form of treatment : suddenly , he seized the collector s wrist and dragged him across the ward to a mattress on which , pale as milk beneath a cloud of flies , a gaunt man lay shivering , stark naked . the collector offered no suggestions so the doctor explained that he had used the best treatment known to medical science the treatment which , for want of a specific , every physician worthy of the name accorded his cholera patients calomel , opium and poultices . ( 165166 ) suddenly , he seized the collector s wrist and dragged him across the ward to a mattress on which , pale as milk beneath a cloud of flies , a gaunt man lay shivering , stark naked . the collector offered no suggestions so the doctor explained that he had used the best treatment known to medical science the treatment which , for want of a specific , every physician worthy of the name accorded his cholera patients calomel , opium and poultices . ( 165166 ) farrell s text echoes almost precisely reports from two sources : firstly , the medical times & gazette of 1854 , mentioned explicitly in his handwritten notes , and secondly , a report entitled general board of health report on the different methods of treatment pursued in epidemic cholera in the provinces throughout england and scotland in 1854 published in 1855 by the royal college of physicians . the first of these sources contains a number of articles and reports which detail contemporary debates , treatments and publications on cholera , including case histories of outbreaks in 1832 , in london in 1849 and in newcastle and london again in 1853 , all of which are reflected in farrell s novel . more precisely , much of dunstaple s approach to treating cholera is drawn from an article entitled statistics of the cases of the cholera epidemic 1853 by j. s. pearse and jeffrey a. marston . pearse and marston assert that the immediate treatment of cholera should involve a warm bath , a blister to the spine and calomel ( medical times and gazette 1854 , 130 ) ; dunstaple , when arguing with mcnab , states that we must consider means of counter - irritating the disease hence a warm bath perhaps , and a blister to the spine ( farrell 2007 , 254 ) . through his repetition of and adherence to such methods of treatment , quack, his investigation of native medicine such as the use of burtunga ants to close wounds acting as evidence of his inability to practice ( 94 ) . farrell s exactitude in dunstaple s description of acceptable treatment for cholera suggests that on the different methods of treatment pursued in epidemic cholera along with other contemporary publications of a similar nature may well have also comprised part of his original research . on the different methods of treatment pursued in epidemic cholera written by drs . paris , alderson , babington , tweedie and bagshaw - ward of the general board of health and part of the treatment committee of the medical council outlines four main treatments for cholera infection : alterative , astringent , stimulant and cathartic , concluding that calomel and opium stand highest in success when used as part of the astringent method ( paris et al . dunstaple again echoes almost exactly the report s assertion that cholera attacks the mucous lining of the intestinal canal but instead of using calomel and opium in the astringent method , he instead combines elements of alterative and stimulant forms , using chloroform and turpentine along with mustard - coated flannels . incidentally , the medical board s report concludes that the alterative and stimulant forms of treatment to have a 36.2% and 54% rate of death respectively . to put these statistics into perspective , they are nearly double and triple the death rate of the recommended treatment . not for the first time , farrell illustrates a gulf between the good intentions and the ill - effects of imperial methods . farrell later develops the dispute between dunstaple and mcnab from one of the initial disagreements over appropriate treatment into an open conflict over the miasma and water - borne theories of cholera . in the latter of two scenes , appropriately set in the garrison s church as a means of emphasising and undermining the notions of faith and belief in both science and the british christian mission , dunstaple demands that mcnab justify his hydration - based treatment of cholera patients . in this exchange , dunstaple repeats his assertion that impure air is the only cause of cholera infection : dr baly finds the only theory satisfactorily supported by evidence is that which regards the cause of cholera as a matter of some process , whether chemical or organic , in impure or damp air dr dunstaple paused triumphantly for a moment to allow the significance of this to seep in ( 252 ; emphasis in original ) . farrell again uses period sources , basing dunstaple s arguments on the report on epidemic cholera by drs . william baly and william gull , royal college of physicians ( 1854 ) , referenced in both the medical gazette of 1854 and the report by drs . paris et al . , and mcnab s ripostes on the research undertaken by john snow in london between 185354 . despite employing the weight of snow s statistical analysis of the 1853 cholera epidemic , mcnab s counter - argument fails to convince the garrison of krishnapur : with the best will in the world and in ideal circumstances it is next to impossible to escape cerebral indigestion as someone quotes comparative figures as fluently as dr mcnab ( 266 ) . the statistically - sound analyses and alternative forms of treatment conducted by snow in london are shown to gain little traction in an indian context ; in this sense , farrell s novel is reflective of the fiercely divided state of the indian medical service ( ims ) , which continued to support miasma theory well into the early twentieth century , arguing that environmental pollution , bad air , poor sanitation as well as impure water were the source of cholera in india ( klein 1980 , 30 ) . mark harrison argues that snow s analysis asked only a limited range of questions and excluded a number of factors pertinent to india and colonial space ( 2002 , 191 ) . james bryden , maintained that there were also distinct atmospheric and meteorological differences between britain and india which affected the transmission and treatment of cholera ( 1869 , 14 ) . e. m. collingham notes that european ideas and their proponents were not often afforded credibility by an ims dominated by an older generation of doctors with out - moded concepts of the body and its treatment ( 2001 , 9091 ) . for instance , in the 1870s , the indian sanitary commissioner j. m. cunningham condemned snow s ideas as mere hypothesis based on inexact and imperfect evidence ( klein 1980 , 31 ) ; in krishnapur , dunstaple similarly derides mcnab s convictions as rubbish and charges mcnab as unable to provide proof of his theories beyond these statistics . denying the analyses of mcnab and by extension snow , the garrison instead take comfort from the authority associated with empire , again drawing comparison to farrell s contemporary moment in which he suggests that the british public are likewise denying the evidence before them and are instead looking backwards to the perceived securities of the past . the point of farrell s faithful use of his source material here is explored in john spurling s essay entitled as does the bishop. spurling calls attention to the way in which farrell s choice of medium , the historical novel , allows him to observe human nature more coolly and clearly , from a seat in the gods, more often than not through the use of dramatic irony ( 1993 , 155 ) . the immediate joke here is that for all dunstaple s passionate conviction and denunciation of the water - borne theory , the reader knows that miasmatic transmission of cholera is eventually disproven and that history will vindicate mcnab . the use of contemporary medical journals as the basis for dunstaple and mcnab s dialogue again reveals the layering and nuance present in the author s use of reciprocity and exchange particular to his historical novels , both in terms of literary form and theme . instead of simply employing it as a plot point to illustrate tension , farrell uses the cholera debate as a lens through which to satirise further the adherents of empire in both victorian society within the novel and contemporary society outside of it . as critics have observed , when farrell plays off the present against the past he is critical of both ( goonetilleke 2003 , 412 ) . here , farrell s split perspective on cholera seeks not only to highlight the divisions inherent to empire both past and present , but also to draw a parallel with contemporary debate on how best to treat the metaphorical ills of the nation either through reliance on traditional , if not entirely successful methods , or by disregarding the past and embracing something new and unproven . farrell takes this reliance on tradition he views as endemic to the british character to ever more ridiculous extremes in order to illustrate both its redundancy in a modern context and the hollowness of its prestige . for example , when dunstaple again explains the proper methods by which cholera should be treated he does so with revealing grandiloquence : [ t]o relieve the pains in the head we might order leeches to the temples . an accepted method of counter - irritation in cholera is with sinapisms applied to the epigastrium or , if i must interpret these learned expressions for the benefit of my distinguished colleague , with mustard plasters to the pit of the stomach ( 254 ) . though the application of leeches and poultices appear archaic , dunstaple s treatments were widely adhered to in contemporary colonial medicine ; in a moment of relief in maria germon s journal of her experiences at lucknow , she notes with joy that her husband charlie brought over 6 bottles of mustard as we had very little and it was in great demand in cases of cholera ( 1870 , 56 ) . moreover , the combination of leeches and poultices illustrate the central paradox of farrell s novel and the victorian age he criticises , namely that the actions of its inhabitants illustrate faith in a world governed by logic and reason while exercising neither themselves ; their enlightened principles are lost in a desire to retain their position . farrell intimates that a preference for tradition against all better judgement is equally applicable in a 1970s context , as he stated in his interview with dean : i hoped to say something about how we , in our thriving modern world of the 1970s hold our own ideas ( dean 1973 , 12 ) . by making his colonial victorians ridiculous , farrell strives not just to lampoon them and their views but also to make his contemporary audience consider how their own actions and judgments may one day too be assessed . the unsettling effect of the oscillation between tradition and an uncertain alternative is keenly illustrated by farrell . a source of much comedy during the debate over the treatment of cholera is the fact that the garrison took to carrying cards in their pockets which gave the relevant instructions in case they should find themselves too far gone to claim the doctor they wanted ( 249 ) . as both doctors hold forth on their respective theories , various members of the garrison cross - out and rewrite their preferred choice of doctor multiple times ( representative of the constant swinging back and forth in opinion on medical treatment ) . farrell captures a picture of britons , to paraphrase homi bhabha , caught uncertainly in the act of composing themselves , indecisively caught between their perception of comfort and familiarity in tradition and the hard logic of reason and fact ( 2005 , 70 ) . farrell expands his metaphor to the doctors themselves , describing dunstaple as a kindly and paternal man possessing authority and good humour the more experienced , and hence more reliable of the two ( 250 ) . seldom smiled and seemed to take a pessimistic view of your complaint , whatever it was ( 250 ) . farrell suggests that , quite understandably , the familiar , paternalistic view of empire is preferable to the new , disquieting prospect of a post - imperial britain , allowing one of his characters , the magistrate , to observe with knowing glee ( h)ow much more easily they were swayed by prestige than by arguments! ( 253 ) . however , farrell further intimates that to continue to accept the rose - tinted , traditional view of empire is a dangerous as belief in the effectiveness of mustard poultices as a cure for bacterial infection . as if to finally underscore the destructive potential of such passionate commitment to the past , farrell has dunstaple swallow a bottle of rice - water discharge gathered from a cholera patient in a bid to disprove mcnab once and for all . in another example of deliberate anachronism akin to his mention of moving daguerreotypes , the episode in which dunstaple deliberately drinks rice - water discharge is in reference to the near identical actions of dr . an outspoken critic of robert koch and an opponent of bacteriology , von pettenkofer s consumption of cholera bacilli proved an equally misguided act of defiance in the face of all evidence to the contrary ( klein , 1980 , 45 ) . however , von pettenkofer s and dunstaple s actions nonetheless serve as illustrative examples of the power of conviction and the longevity of tradition in times of rapid change . alongside the mounting tensions over the treatment of cholera , another medical thread running through the novel is the veracity or otherwise of phrenology . franz joseph gall of vienna , phrenology was fashionable for the early part of the nineteenth century , particularly between the 1820s and 1840s ( 13 ) . although described as a pseudo - science by reputable medical journals in the 1840s , the fashion for phrenology and belief in its principles remained popular throughout the nineteenth century and , in some instances , into the early twentieth ( hanen et al . phrenology , like cholera , becomes a similarly vital means by which farrell is able to illustrate the manner in which outdated medical ideas were able to retain a degree of professional cachet in a colonial setting long after their abandonment in european medical discourses . the inclusion of phrenology enables farrell not only to satirise british convictions in constant scientific and medical progress but also the actions and opinions of those who propagate such beliefs without recognition of their redundancy . also , and perhaps more importantly , phrenology becomes one of the few instances in which farrell s depicts indian characters in any detail other than as thinly drawn antagonists . as mentioned above , the siege of krishnapur , focuses mainly on the coloniser ; the discussion that surrounds phrenological practice within the novel becomes a means of providing native characters with a more prevalent fictive voice , though , crucially , not in a language or context of their own . phrenology is another european import to colonial space and more readily associated with control as part of the classificatory discourse of the coloniser ; according to lawrence james , phrenology was used in india to catalogue criminal tribes ( james 2007 , 202 ) . upholding this association with authority , phrenology s greatest proponent among the garrison in the siege of krishnapur is mr . the novel contains repeated mentions of the magistrate s devotion to phrenology , whilst farrell s original manuscripts also include numerous extended sections or deletions from the finished text . though apart from the magistrate phrenology is largely discounted by other residents of krishnapur , the fact that the subject remained popular amongst colonial practitioners of the period is clear , as the journal leaves turned down or the autobiography of an indian army surgeon ( 1854 ) reveals . in this account , the anonymous army surgeon author confesses himself to be a devotee of phrenology and decides to conduct an investigation on scientific principles on a hindu thugee prisoner reputed to have killed approximately 1,100 men over a period of fifty years ( 1854 , 126 ) . the magistrate experiences a similar desire to get to grips with the skulls of his compatriots and of indians alike ; for instance , during a discussion about evacuating the garrison the magistrate was surveying the behaviour of the cantonment in an objective and utterly scientific manner would there be a common skull - form that rejected it ? he hungrily eyed heads , even neglecting his work to haunt the balder members of the cantonment ( farrell 1972 , 102103 ) . farrell uses phrenology to satirise a number of victorian concerns , particularly those related to the expression of female sexuality and the perceived effect of colonial space on morality ( ghosh 2006 , 50 ) . for example , the magistrate fixates his attentions on the so - called fallen woman of the garrison , miss lucy hughes , recently jilted after an affair with an army officer . convinced that lucy s organ of amativeness was extraordinarily well developed, namely the part of the brain alleged to control sexual desire , farrell describes how the magistrate had been in the position of a scientist who has made a discovery which he knows to be true but is unable to prove ( 241 ) . amativeness appears to have been integral to farrell s decision to include phrenology in his novel ; of the five index cards on phrenology amongst his papers , one is devoted entirely to notes and descriptions of amativeness . chancing upon lucy alone , the magistrate finally makes his move just after the siege is relieved . in a reversal of a traditional romantic denouement expected of an imperial adventure novel : the magistrate put a companionable hand on her shoulder and then , after a moment s hesitation , slipped it onto the back of her neck . perhaps lucy would have melted weakly into his bony arms had not an expression of dismay and incredulity come over his face . she promptly slapped him as hard as she could , which was not very hard . she did not know what the matter was but knew instinctively that this was the right thing to do ( 310 ) the magistrate put a companionable hand on her shoulder and then , after a moment s hesitation , slipped it onto the back of her neck . perhaps lucy would have melted weakly into his bony arms had not an expression of dismay and incredulity come over his face . she promptly slapped him as hard as she could , which was not very hard . she did not know what the matter was but knew instinctively that this was the right thing to do ( 310 ) beyond the comedy to be found in the play on lucy s natural and social instincts , the magistrate s shock at finding his principles challenged echoes the situation experienced by the surgeon in leaves turned down . during his exchange with the thugee prisoner best of bumps ; this throws him into some turmoil , leading him to doubt his phrenological devotion : that night , phrenology and i had a strong tussle ; and she had much difficulty in re - establishing herself in my estimation for this specimen of farrell uses phrenology as a means of exposing the hollowness of british convictions . in direct transposition of the wider conflict of the novel , farrell equates the magistrate s ardent , if erroneous , belief in phrenology with belief in the benefits of british rule to india . the magistrate s discovery and flight indicates , at least initially , either a realisation of the shortcomings of phrenology and with it the british claim on medical and civilising authority or even a sense of horror at what his investigation reveals . however , farrell s novel is ambiguous and does not offer conclusive proof that the magistrate is forced to re - evaluate his beliefs . again , much like in leaves turned down where the anonymous author convinces himself that phrenology was not at fault after all , farrell s manuscript fragments suggests the same possibility with regards to the magistrate : [ t]he magistrate was aware that it was not phrenology that was in error , but his own inadequate use of it ( farrell 1972 , 70 ) . though the magistrate is surprised by his discovery , there is no concrete suggestion that he will re - evaluate his convictions . of course , the british presence in india continued long after the mutiny was put down , dissolving the east india company and instead reinforced as a crown endeavour . farrell illustrates the resilience of long - held beliefs and the longevity of their consequences , even after compelling evidence to disprove them . in his decision to delete this section from the published version , however , farrell makes the ambiguity inherent to the ending of the siege of krishnapur potentially hopeful , suggesting that even the strongest beliefs are subject to change if given the necessary impetus . farrell s ambiguity runs deeper when considered in relation to hari , the maharajah s son imprisoned by the collector throughout the siege and one of few native characters seen up close or given dialogue . imprisoned allegedly to ensure his safety as well as for his value as a hostage , farrell characterises hari as susceptible to british instruction ; he is first presented eating a boiled egg whilst reading blackwood s magazine ( 71 ) and represents the class of anglicised indians encouraged by thomas babington macaulay s minute on education in india : a class of persons , indian in blood and colour , but english in taste , in opinions , in morals , and in intellect ( jayapalan 2005 , 56 ) . hari s characterisation is significant for its evocation of mimicry and embodied racial difference . the chapter in which fleury meets hari and is given a tour of the palace is focalised through fleury , allowing farrell to present and critique the latent racism of the supposedly enlightened coloniser . fleury notes with disconcertion that both hari and the paintings of the previous maharajahs that adorn the palace walls are really just one face repeated again with varying skill ( 71 ) . fleury here repeats and rearticulates the prejudices of the british garrison , revealing both the tendency to homogenise the figure of the indian and also the extent to which mimicry acts as an unsettling force upon the coloniser . as bhabha argues in the location of culture , an image , and by extension here hari himself , identical by virtue of the difference that defines it ( 2004 , 106 ) . hari is thus left paradoxically with and without form ; his identity another site of contestation between british and indian control . farrell s inclusion of a character such as hari affirms morey s suggestion that the relationship between coloniser and colonised is configured as a site of unequal exchange ( 2000 , 11 ) . hari s imprisonment ensures that the maharajah s forces do not attack ; whilst the british improve their own position , it is at the cost of hari s liberty . farrell takes the idea of unequal exchange further , however , through hari s interactions with the magistrate . during his captivity , hari is given a book on phrenology by the magistrate and becomes a devotee of what he calls frenloudji science of head, practicing his new found discipline on his prime minister , who has been taken into captivity with him ( 177 ) . hari too is absorbed by the study of amativeness , stating that amativeness the faculty gives rise to sexual feeling not very powerful organ in prime minister . in me , it is fearfully , fearfully powerful so that all other organs wither away ( 177178 ) . farrell illustrates how phrenology is used to classify and construct the figure of the native ; hari confirms and labels himself under the stereotype of the sexually licentious oriental ( said 2003 , 4 ) . the irony inherent to farrell s portrayal of hari is that in a novel whose characters are convinced of the good they are bringing to india , the only thing that the native receives in the course of his contact with the british is his devotion to frenloudji; the much lauded , so - called civilising principles of empire instruct hari in a falsehood and serve only to make him complicit in his own denigration ( 177 ) . beneath the surface comedy of hari s pidgin english , itself double - edged , farrell s inclusion of phrenology in his novel represents a powerful indictment of the british empire in india and the contemporary legacy of imperial power . phren from the greek for mind and logos as language , represent two elements of both history and contemporary modernity with which farrell seeks to engage . indeed , farrell invites the reader to investigate the minds of his characters as they investigate those of others ; phrenology becomes another of farrell s means of examining and satirising the mind of the imperial briton and the way that mind expresses not only its hopes , desires and fears but also its perception of itself and the world around it through fiction . moreover , by highlighting the shortcomings of victorian and imperial values , farrell seeks to counter any nostalgia for empire in the post - imperial 1970s . by making such an obvious example of pseudoscience a dominant theme in the novel , farrell s subversion of victorian scientific and medical progress and the civilising principles of empire is clear ; for farrell , the only lasting effects that the great beneficial disease of british imperialism leaves on the indian body are bullets , subjugation and quackery . though his choice of medical matters is varied , the disputes over phrenology and cholera are united by farrell s central narrative intention , namely to strike at the heart of what umberto eco identifies as endoxa , the accepted understanding of values and morals as propagated by dominant culture ( 1979 , 161 ) . by interlinking postmodern and postcolonial concerns within the format of the historical novel , farrell is able to engage parodically with the history of british colonial india as a means of exploring the contemporary crisis of englishness provoked by the failure of empire after the second world war ( crane and livett 1997 , 99 ) . rather than the received historical narrative of british unity in the face of external assault , in this instance the mutinous sepoys , farrell recasts the krishnapur garrison as inherently divided . by doing so , he posits that the disintegration of empire is not a uniquely contemporary phenomenon but an historical one . the dramatic irony farrell uses at a surface level gives way to a more tragicomic mode of transmission , one in which the reader is aware that the efforts of the garrison , indeed the efforts of empire , will one day count for little . it is telling that during one chapter when the collector turns his mind to what empire may be like a hundred years hence in 1957 , farrell describes his usually formidable mental powers as suddenly enfeebled . by weaving factual source material into the fictional fabric of his novel , farrell is using the historical novel to play complex games with time and place , position and perspective . the enjoyment for farrell s readership is in their knowledge that phrenology , along with the airborne theory of cholera transmission , is debunked in time ; however , his characters remain largely convinced of their validity . by using medicine in this way , farrell is able to turn the satire back to face his contemporary audience , and by mocking something such as the misguided adherence to the pseudo - science of phrenology , farrell is inviting his readers to examine more closely what contemporary history in the 1970s was doing to the record of british imperialism and its potential effect on their own adherences or beliefs . moreover , it is significant that farrell s novel offers no resolution in the dispute over either cholera or phrenology ; dr . dunstaple dies inconclusively of a heart attack whilst the magistrate and hari are not seen again after their respective exits from the narrative . again , he draws a parallel between the past and the 1970s present in that there are no certainties and that no definitive endings will be forthcoming . coetzee , peter carey and , latterly , hilary mantel.farrell had begun work on a fourth novel by the time of his death , posthumously published as the hill station in 1981 . set in india in 1871 and featuring many of the characters from the siege of krishnapur , it effectively makes empire trilogy into a series . however , because of its incomplete nature , it is not always considered canonical by farrell scholars.the term mutiny is used throughout this piece in reflection of its contemporary lexical currency at farrell s time of writing and not in any opposition to its modern one . the mutiny of 1857 is now largely referred to as the rebellion of 1857 or the first war of independence.see lavinia greacen s j. g. farrell : the making of a writer ( london : bloomsbury , 2000 ) for further detail on farrell s early life.francis b. singh s article , progress and history in j. g. farrell s the siege of krishnapur ( chandrabhaga 2 1979 , 2329 ) notes the generally poor presentation of native subjects in the novel and as such does not categorise farrell as postcolonial.farrells first instalment of empire trilogy is of course called troubles ; a title with a particular relevance and resonance in the 1970s . intending to continue this trend , difficulties ; see a. johnson , ghosts of irish famine in j. g. farrell 's the siege of krishnapur the journal of commonwealth literature , vol . his 1973 interview with malcolm dean , farrell expressed how he had thought of constructing the novel entirely from contemporary nineteenth - century insights and observations ; dean , an insight job, p. 11.germon fever in july of 1857 , though it is strongly implied by the editors of his diaries that it was most likely cholera that killed him , a fact later corroborated by g. w. forrest s a history of the indian mutiny reviewed and illustrated from original documents , vol . i ( london : william blackwood & sons , 1904).i would like to acknowledge that my own inspection of farrell s papers was made possible through generous support from a wellcome trust small grant ( grant number : 100559/z/12/z ) awarded in october 2012 and the kind permission of trinity college library , dublin.dr . baly directly ; it is also referenced in the article by paris et ai and reproduced in the medical gazette of 1854 referred to in farrell s papers.dr . dunstaple also asks fleury if he would fetch half a dozen bottles of mustard during the novel ( farrell 2007 , 161 ) . fallen woman referred to in reverend polehampton s diary ( polehampton 1858 , 108109 ) . for a book about india , indians themselves are woefully under represented in the novel , something farrell later acknowledged with regret . 1957 has a personal resonance for farrell as well as a thematic one ; the choice of year deliberately recalls the aftermath of the suez crisis , the widely accepted death - knell of empire , and was also the year during which farrell suffered his bout of polio . farrell had begun work on a fourth novel by the time of his death , posthumously published as the hill station in 1981 . set in india in 1871 and featuring many of the characters from the siege of krishnapur , it effectively makes empire trilogy into a series . however , because of its incomplete nature , it is not always considered canonical by farrell scholars . the term mutiny is used throughout this piece in reflection of its contemporary lexical currency at farrell s time of writing and not in any opposition to its modern one . the mutiny of 1857 is now largely referred to as the rebellion of 1857 or the first war of independence. see lavinia greacen s j. g. farrell : the making of a writer ( london : bloomsbury , 2000 ) for further detail on farrell s early life . francis b. singh s article , progress and history in j. g. farrell s the siege of krishnapur ( chandrabhaga 2 1979 , 2329 ) notes the generally poor presentation of native subjects in the novel and as such does not categorise farrell as postcolonial . farrell s first instalment of empire trilogy is of course called troubles ; a title with a particular relevance and resonance in the 1970s . intending to continue this trend , the siege of krishnapur was originally entitled difficulties ; see a. johnson , ghosts of irish famine in j. g. farrell 's the siege of krishnapur the journal of commonwealth literature , vol . 46 , ( new york : sage , 2011 ) , pp . 275292 . in his 1973 interview with malcolm dean , farrell expressed how he had thought of constructing the novel entirely from contemporary nineteenth - century insights and observations ; dean , an insight job, p. 11 . fever in july of 1857 , though it is strongly implied by the editors of his diaries that it was most likely cholera that killed him , a fact later corroborated by g. w. forrest s a history of the indian mutiny reviewed and illustrated from original documents , vol . i would like to acknowledge that my own inspection of farrell s papers was made possible through generous support from a wellcome trust small grant ( grant number : 100559/z/12/z ) awarded in october 2012 and the kind permission of trinity college library , dublin . baly directly ; it is also referenced in the article by paris et ai and reproduced in the medical gazette of 1854 referred to in farrell s papers . . dunstaple also asks fleury if he would fetch half a dozen bottles of mustard during the novel ( farrell 2007 , 161 ) . fallen woman referred to in reverend polehampton s diary ( polehampton 1858 , 108109 ) . for a book about india , indians themselves are woefully under represented in the novel , something farrell later acknowledged with regret . 1957 has a personal resonance for farrell as well as a thematic one ; the choice of year deliberately recalls the aftermath of the suez crisis , the widely accepted death - knell of empire , and was also the year during which farrell suffered his bout of polio .

this article examines j. g. farrell s depictions of colonial medicine as a means of analysing the historical reception of the further past and argues that the end - of - empire context of the 1970s in which farrell was writing informed his reappraisal of imperial authority with particular regard to the limits of medical knowledge and treatment . the article illustrates how in the siege of krishnapur ( 1973 ) , farrell repeatedly sought to challenge the authority of medical and colonial history by making direct use of period material in the construction of his fictional narrative ; by using these sources with deliberate critical intent , farrell directly engages with the received historical narrative of colonial india , that the british presence brought progress and development , particularly in matters relating to medicine and health . to support these assertions the paper examines how farrell employed primary sources and period medical practices such as the nineteenth - century debate between miasma and waterborne cholera transmission and the popularity of phrenology within his novels in order to cast doubt over and interrogate the british right to rule . overall the paper will argue that farrell s critique of colonial medical practices , apparently based on science and reason , was shaped by the political context of the 1970s and used to question the wider moral position of empire throughout his fiction .

Cultural and critical contexts Dont drink the water cholera Bringing matters to a head phrenology Conclusions Endnotes

PMC4451371

even small changes in glucose metabolism may contribute to the onset of cardiovascular disease and endothelial dysfunction due to peripheral insulin resistance . acute myocardial infarction affects a large proportion of patients with dm2 ; thus , the evidence of subclinical atherosclerosis would be helpful for preventive strategies in these individuals [ 1 , 2 ] . even knowing that hyperglycemia is one of the ways that can lead to the development of atherosclerosis , the mechanisms underlying coronary artery disease ( cad ) in individuals with type ii diabetes are not known for sure . thus , besides serum glucose , other markers related to the pathophysiology of cardiovascular diseases might also be evaluated . recent prominent candidates for prediction of cad in dm2 patients include thiols ( cysteine , homocysteine , methionine oxidized glutathione , and reduced glutathione ) , n - acetyl--d - glucosaminidase ( nagase ) , endogenous nitric oxide synthase inhibitors ( adma ) , nitrate + nitrite ( nox ) , nitrotyrosine , and s - nitrosothiols ( rsno ) . hence , the purpose of this study was to identify important biochemical alterations that could distinguish dm2 individuals with and without cad . in this study , 96 volunteers were enrolled and classified into the following groups : type 2 diabetes mellitus ( dm2 , n = 21 ) and type 2 diabetes mellitus with coronary artery disease ( dm2 + cad , n = 75 ) ( table 1 ) . volunteers of both genders ( aged 21 years ) were screened at the following brazilian institutions : instituto dante pazzanese de cardiologia , irmandade da santa casa de misericrdia de so paulo , and faculdade de cincias mdicas da universidade metodista de santos . all subjects were fully informed about the details of the study and protocol and provided written informed consent . dm2 was defined according to the american diabetes society criteria , whereas participants with coronary artery disease were defined as patients who had acute myocardial infarctions confirmed by ecg , laboratory exams , and clinical symptoms . exclusion criteria used were pregnancy , psychiatric disorders , renal and/or hepatic diseases , smoking , alcoholism , cancer , or other pathological conditions that could interfere with the study . venous blood was collected in tubes with and without edta after 12 hours of fasting . the concentrations of fructosamine , glycated hemoglobin ( hba1c ) , glucose , total cholesterol , triglyceride , high - density lipoprotein- ( hdl- ) cholesterol , and very low density lipoprotein- ( vldl- ) cholesterol were estimated by enzymatic methods using kits ( biosystems sa , barcelona , spain ) . the friedewald equation was used to calculate the low density lipoprotein- ( ldl- ) cholesterol . n - acetyl--glucosaminidase activity was determined based on the methodology described by reglero and cabezas ( 1976 ) . 100 l of plasma was added to 1.25 ml citrate buffer ( 0.1 m , ph 4.4 ) and incubated at 37c with 0.25 ml 0.01 m p - nitrophenyl - n - acetyl - glucosaminide in citrate buffer for 15 minutes . the reaction was interrupted with 1.5 ml of sodium carbonate ( 0.2 m , ph 10.4 ) and the final product was measured by spectrophotometer at 405 nm . one unit of enzyme was defined as the amount released 1 mol p - nitrophenol / min . we used the nitric oxide analyzer ( noa , sievers , usa ) based on the chemiluminescence reaction between nitric oxide and ozone . the reduction of nitrate and nitrite ( nox ) with vanadium chloride was used to convert nox to oxide nitric . calibration curves with multiple levels were performed with an external standard ( sodium nitrate ) using the bag program software ( version 2.2 , sievers , usa ) . the samples were extracted with cold ethanol ( 0.5 ml sample and 1.0 ml of ethanol at 0c ) . after vortexing , the solution was stored on ice during 30 minutes and then centrifuged at 9000 g for fifteen minutes . the standard s - nitroso - albumin ( sno - alb ) was used to quantify total s - ntrosothiol serum . the synthesis was obtained by the reaction of nitrite with human albumin in 0.1 m hcl . this was incubated in the dark for 2 hours and then the absorbance was measured at 336 mm ( = 3874 mcm ) . the calibration curve was made with 10 , 50 , 100 , 250 , and 500 nm sno - alb . 1.0 ml of plasma was added to 10 l of n - ethylmaleimide ( 500 mm ) . after homogenizing , 100 l of sulfanilamide ( 1% ) was added and homogenized again . we used the equipment nitric oxide analyzer ( noa , sievers , usa ) and injected 500 l of sample and 500 l of standard . the reaction solution was composed of 8 ml of glacial acetic acid , 2 ml of ki ( 50 mg / ml ) , 300 l of decanol , and 200 l of cuso4 ( 200 mm ) at 70c . plasma concentrations of adma , sdma , and l - arginine were determined by the technique of capillary electrophoresis ( biofocus 2000 , bio - rad laboratories , inc . ) . the blood was collected with edta and centrifuged at 1,000 g for 10 minutes at 4c to obtain plasma . the calibration curve was performed by adding the standard of adma , sdma , and l - arginine in pooled plasma . samples and standard were precipitated with ethanol , centrifuged at 9.000 g for 15 minutes at 4c , and then derivatized with fluorescein 5-isothiocyanate . the injection was done under pressure ( 1 psi.sec ) and the race was performed in a fused silica capillary ( 85 cm length and 50 internal diameter m ) at 20 kv . running buffer consisted of 50 mm boric acid and 20 mm 3-cyclohexylamino-1-propanesulfonic adjusted to ph 10.8 . the detector laser - induced fluorescence ( biofocus lif , bio - rad laboratories , inc . ) operated at 488 nm ( excitation ) and 520 nm ( emission ) ( causs et al . the blood was collected with edta and centrifuged at 1000 g for 10 minutes at 4c to obtain plasma . in 200 l in plasma 2 l internal standard ( 1 mm n-(2-mercaptopropionyl ) glycine ) and 20 l tri - n - butylphosphine 10% ( v / v dimethylformamide ) were added . then , 200 l 10% trichloroacetic acid containing 1 mm edta was added . after homogenizing samples , they were centrifuged at 13.000 g for 15 minutes at 4c . in 100 l of the supernatant was added to 100 l 0.5 m phosphate buffer and adjusted to ph 7.5 with 1 m na3po4 . the derivatizer ( 5-bromomethylfluorescein ) was added to the samples at a molar ratio of 5 to 10 times excess and the preparations are incubated for 15 minutes at 60c . after derivatization , the sample was diluted at 1 : 10 with phosphate buffer 0.25 m ph 7.6 and injected with a pressure of 0.5 psi for 1.5 seconds on capillary ( 70 cm length and 50 m internal diameter ) . the capillary electrophoresis instrument ( biofocus 2000 , bio - rad laboratories , inc . ) the detector laser - induced fluorescence ( biofocus lif , bio - rad laboratories , inc . ) operated in 488 nm ( excitation ) and 520 nm ( emission ) . for each run we used the polyclonal anti - nitrotyrosine antibody ( upstate , catalog number : 06 - 284 ) . the standard used was nitrated bovine albumin ( nitro - albumin ) prepared by alkaline addition of 1 mm peroxynitrite and 1 mm albumin . the concentration of the nitro - albumin was determined using the molar extinction coefficient of 4300 mcm at 438 nm and ph 9.0 . the plate was sensitized with 0.05 ug nitro - albumin per well and then washed , blocked with milk protein , and rinsed again . the following was added to the plate anti - nitrotyrosine antibody and sample / standard . after incubation , the plate was washed and added to the peroxidase - conjugated antibody ( stressgen biotechnologies corp . ) . after incubation and washing , 2.3 mm luminol and 0.9 mm p - iodophenol ( 200 ul / well ) and 3.9 mm hydrogen peroxide ( 50 ul / well ) were added . the reading of the chemiluminescence produced was immediately performed ( lumicount , packard , meriden , usa ) . the present nitrotyrosine concentrations in the sample were estimated by using the calibration curve and nitrated albumin was expressed as equivalent nitro - albumin . data were expressed as means standard deviation ( sd ) , median ( interquartile range ) , or counts ( percentage ) when appropriate . for univariate analyses , groups were compared by the t - test for approximately normally distributed variables or by means of the mann - whitney u - test for variables with skewed distribution . in addition , we constructed multiple logistic regression models using the backward stepwise selection procedure to ascertain predictors for cad in dm2 patients ( coding scheme : dm2 + cad = 1 , dm2 = 0 ) . variables were sequentially removed from the full model ( all regression terms included ) when the correspondent p value was higher than 0.10 . in order to assess the model performance , we did not employ k - fold partitioning due to the relatively low number of subjects . instead , we used the resubstitution approach , in which the same data are used for both training and testing . for these analyses , each participant has an estimated probability of being dm2 + cad , which is calculated from the logistic model - derived equation based on the participant 's own variables . if the calculated probability was 50% , the subject was assigned as dm2 + cad or dm2 only , otherwise . we quantified model performance by using two measures of classification accuracy that are not susceptible to class imbalance : balanced accuracy ( ba ) and normalized mutual information ( nmi ) . ba is an accuracy measure that takes into account both sensitivity and specificity of the models and is calculated as the average of sensitivity and specificity . nmi ranges from 0 to 100% and is interpreted as the amount by which the examined model reduces one 's uncertainty about the true state of the participants ( e.g. , 0% means that the status is independent of the studied explanatory variables , while 100% suggests that the model fully predicts the status for each individual ) . permutation was used to construct empirical null distributions ( 2,000 shuffles ) in order to compute the statistical significance of both ba and mni measures . the hypothesis of a significantly better fit for the model with biochemical biomarkers plus classical variables for cad ( full model ) compared to the simpler models ( nested models ) was tested by means of a likelihood - ratio test . this test assumes the form = 2(l0 l1 ) , where l1 and l0 are the natural log - likelihoods under the full and nested models , respectively . we used permutation to test whether the full model fits the data significantly better than simpler models by comparing the observed test statistic to those obtained in 2,000 randomly generated shuffles . all data analyses were performed using the stata package ( version 11.0 , stata corp . , college station , tx , usa ) . all calibration curves were linear with a coefficient of determination ( r ) 0.98 . the quantification limit ( 10 : 1 ; signal : noise ) by capillary electrophoresis ( adma , sdma , and l - arginine : 25 nm ; thiols : 50 nm ) and nitric oxide analyzer ( nox and rsno : 10 nm ) were suitable for detection of the analytes in the samples . the elisa also showed a quantification limit adequate for detection of nitrotyrosine ( 30 nm ) . the main demographical , clinical , and biochemical characteristics of the studied participants are shown in table 1 . twenty - one ( 22% ) out of 96 participants with dm2 were classified as dm2 + cad . age , body mass index ( bmi ) , and gender proportion were comparable between dm2 + cad and dm2 groups . however , in an exploratory ( unadjusted ) analysis , levels of methionine , ldl / hdl ratio , n - acetyl--glucosaminidase , and nitrite plus nitrate ( nox ) were significantly higher in dm2 + cad . in addition , levels of l - arginine : adma ratio , fructosamine , and reduced glutathione were significantly lower in dm2 + cad patients compared to their dm2 counterparts . after a holm - bonferroni correction for 25 tests , both nox and fructosamine levels remained significantly higher in dm2 + cad participants compared to the dm2 group ( p = 0.02 and p = 0.006 , resp . ) . to determine putative predictor variables for cad in dm2 , we built a multiple logistic regression model with backward elimination . of the 25 variables tested , eight remained in the final model : ldl / hdl ratio , methionine , reduced glutathione ( gsh ) , l - arginine : adma , nox ( nitrate plus nitrite ) , n - acetyl--glucosaminidase ( nagase ) , gender , and bmi ( table 2 ) . on the basis of this model ( hereafter named full model ) , we constructed an equation that was applied to the complete dataset to predict the status of the 96 studied participants . the overall classification accuracy of this model ( resubstitution method ) is presented in table 3 , along with other two competing models including either only classical variables for cad ( i.e. , ldl / hdl ratio , gender , and bmi ; model 2 ) or emerging biochemical markers only ( i.e. , l - arginine : adma ratio , methionine , gsh , nagase , and nox ; model 3 ) . all of the three models significantly predicted cad more often than can be expected by chance , although it can be verified from table 3 that the performance of the studied models ranges from small to moderate . we sought next to assess whether there is an increase in accuracy for prediction models when emerging biochemical markers are used in combination with classical risk factors for cad . in other words , we tested whether simpler models ( models 2 and 3 ) are equally capable of predicting cad in dm2 patients compared to the full model . this is of paramount importance , since there is a potential of overfitting as the number of variables included in the model increases . we observed that the full model fits significantly better the data compared to either model 2 ( p < 0.001 ) or model 3 ( p = 0.004 ) , indicating that there is a statistically significant gain in accuracy when both emerging biochemical markers and classical risk factors are added to the model for cad prediction . for comparison purposes , less conservative measures of diagnostic accuracy ( e.g. , classification error ) also favor the full model over models 2 and 3 ( data not shown ) . we found that individuals with dm2 + cad have several significant metabolic changes related to endothelial dysfunction and oxidative stress compared to dm2 without cad . in our sample , we observed significant alterations in the following variables : hdl - cholesterol , fructosamine , gsh , sdma , l - arginine / adma , nox , and nagase . in addition , we showed that the prediction accuracy for cad in dm2 patients is significantly increased when six emerging biochemical markers are incorporated into the model along with classical risk factors for cad . first , our investigation should be regarded as a predictor finding study only . although we have developed a multivariable prediction model , we did not aim at the development of a model for use in clinical practice to guide patient management . instead , our study should be viewed as a hypothesis - generating study that warrants further confirmation in larger , well - powered investigations with external validation . second , criticism might be directed at the fact that our definition of cad might lack specificity . because cad was classified conservatively , silent cad might be present among a few patients in the dm2 + cad group . however , in our study , misclassification of the cad status is likely to lead to downwardly biased estimates , ultimately leading to less impressive model predictions ( i.e. , more conservative models ) . third , our sampling scheme was based on volunteers and was not designed to be representative of the population . with that said , our results are prone to a survival bias , since there is a reduced likelihood of enrolling patients who died acutely or who are experiencing severe consequences of myocardial infarction . again , these results are likely to lead to more conservative model predictions , since more prominent metabolic alterations are plausible in advanced cad patients . on the other hand , even though we employed permutation to compute statistical significance , our small sample size is prone to overoptimistic results . indeed , there is a clear potential for overfitting and overestimation , because our models were constructed with a large number of biomarkers relative to the number of participants . in another study conducted by our research group we showed that hyperglycemia correlated with increased production of nitric oxide based on the evaluation of concentrations of nitrate and nitrite . endothelial dysfunction is generally defined as impaired endothelium - dependent vasodilation , related to lower no production or bioavailability . reactive oxygen and nitrogen species ( rons ) , such as h2o2 , o2 , no , and onoo , can be produced in blood vessels by certain drugs and pathological conditions and accentuate or induce endothelial dysfunction . rons may reduce no bioactivity by formation of peroxynitrite or by decrease of enzyme or transporter activities by oxidation of their thiols groups . endothelial dysfunction can contribute to the initiation , progression , and clinical manifestations of cad . the increase in s - nitrosothiols may be a consequence of glycation of proteins , which facilitates the nitrosation of protein thiols by nitric oxide . the glycated proteins can act as a sink for nitric oxide because s - nitrosation reduces their competence to release nitric oxide which reflects directly on the pathogenesis of endothelial dysfunction . nitric oxide can react with superoxide anion , which leads to the formation of a potent oxidant , peroxynitrite , and in sequence generation of hydroxyl radicals and nitrogen dioxide [ 18 , 21 , 22 ] . the tyrosine , free or protein - bind , may react with nitrogen dioxide generating nitrotyrosine , which is considered a biomarker of the formation of peroxynitrite . in this present study , only the increase of nox was demonstrated with no differences for nitrotyrosine , even though reduced glutathione and reduced glutathione / oxidized glutathione ratio concentrations were observed . the endogenous nitric oxide synthase inhibitors , represented by symmetric dimethylarginine ( sdma ) and asymmetric dimethylarginine ( adma ) , also play an important role in endothelial dysfunction . in the present study , reduced concentration of l - arginine / adma in dm2 + cad group in comparison with dm2 without cad reinforces the fact that endothelial nitric oxide synthase inhibition occurs along the atherosclerosis process . accordingly , it has been shown that l - arginine / adma levels diminished parallel to the impairment of vasodilatation in dm2 patients after lipid ingestion . moreover , it was previously reported that low l - arginine / adma ratio was not related to either the presence of macroangiopathy or the time of disease manifestation in dm2 patients , but rather to the metabolic control of the diabetes . thus , in the present study , hypercholesterolemia observed in dm2 + cad group could be related to adma increase , occasioning a lower production of no and leading to endothelial dysfunction . however , nitric oxide low production could not be confirmed in this study based only on the increase of nitrite plus nitrate concentration generation . studies have shown a reduction of endothelial dysfunction by rising l - arginine / adma ratio . lipid peroxidation causes peroxidative damage to tissue proteins and may raise the rate of proteolysis . alternatively , there may be a dysfunction or downregulation of di - methylarginine dimethylaminohydrolase , the enzyme that converts adma to l - citrulline . hypercholesterolemia may alter the regulation or function of di - methylarginine dimethylaminohydrolase , thereby resulting in intracellular accumulation of adma . hypercholesterolemia plus hyperglycemia is a factor that contributes to oxidative stress due to autoxidation of glucose and nonenzymatic protein glycation . thiols play an important role on the oxidative processes and therefore may directly or indirectly contribute in endothelial dysfunction . glutathione is a major thiol used as a biomarker of oxidative stress and is found in the forms of gsh ( reduced glutathione ) and gssg ( oxidized glutathione ) . in the present study , the dm2 + cad group showed lower gsh / gssg and gsh concentration , compared with dm2 without cad group . this decrease of reduced glutathione may be due to activation of the polyol pathway in which glucose is reduced to sorbitol by the nadph - dependent aldose reductase [ 30 , 31 ] . this enzyme presents a high michaelis - menten constant for glucose and therefore this metabolic via is only quantitatively significant in hyperglycemia . thus , whenever the nadph / nadp ratio is decreased in hyperglycemic state , it will result in prejudice in the regeneration of reduced glutathione . also as a consequence , the synthesis of nitric oxide by nitric oxide synthase will be impaired due to the nadph dependence [ 31 , 32 ] . apparently , the decrease in gsh concentration has a relation to the presence of atherosclerotic inflammation process in patients with cad . the activity of n - acetyl--d - glucosaminidase ( nagase ) in plasma has been used as a biomarker of endothelial dysfunction . in the present study , nagase activity was significantly higher in dm2 + cad group in comparison with the dm2 without cad group . nagase is a lysosomal enzyme produced by many cells , including not only endothelial cells , but also smooth muscle and kidney . showed correlation between measurements of brachial artery diameter and blood flow via doppler ultrasonography with nagase activity in patients with dac . additionally , nagase activity showed positively correlated with insulin resistance in patients with cad . according to komosiska - vassev et al , the increase of nagase activity can be considered an indicator of the intensity of endothelial cell dysfunction . several studies also demonstrated that nagase is increased in individuals with dm2 , when compared to healthy individuals and that nagase activity is high in individuals with dm2 and previous history of cardiovascular disease ( myocardial infarction ) in comparison with individuals with dm2 without previous coronary events [ 34 , 35 , 37 ] . thus , our data reinforce previous data showing that the increase of nagase activity in individuals with dm2 can be associated with the presence of cad .

we investigated the potential of a panel of 22 biomarkers to predict the presence of coronary artery disease ( cad ) in type 2 diabetes mellitus ( dm2 ) patients . the study enrolled 96 dm2 patients with ( n = 75 ) and without ( n = 21 ) evidence of cad . we assessed a biochemical profile that included 22 biomarkers : total cholesterol , ldl , hdl , ldl / hdl , triglycerides , glucose , glycated hemoglobin , fructosamine , homocysteine , cysteine , methionine , reduced glutathione , oxidized glutathione , reduced glutathione / oxidized glutathione , l - arginine , asymmetric dimethyl - l - arginine , symmetric dimethyl - l - arginine , asymmetric dimethyl - l - arginine / l - arginine , nitrate plus nitrite , s - nitrosothiols , nitrotyrosine , and n - acetyl--glucosaminidase . prediction models were built using logistic regression models . we found that eight biomarkers ( methionine , nitratate plus nitrite , n - acetyl--glucosaminidase , bmi , ldl , hdl , reduced glutathione , and l - arginine / asymmetric dimethyl - l - arginine ) along with gender and bmi were significantly associated with the odds of cad in dm2 . these preliminary findings support the notion that emerging biochemical markers might be used for cad prediction in patients with dm2 . our findings warrant further investigation with large , well - designed studies .

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